MULTIPLEX, SENSITIVE AND RAPID NUCLEIC ACID DETECTION CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit under 35 U.S.C. § 119(d) of the US Provisional Application No. 63/043,853 filed June 25, 2020 and US Provisional Application No. 63/046,448, filed June 30, 2020, the contents of both of which are incorporated herein by reference in their entireties. TECHNICAL FIELD [0002] The technology described herein relates to methods, compositions, kits, and systems for detecting nucleic acids in multiplex format. BACKGROUND [0003] Rapid, low-cost, sensitive methods for point-of-care diagnostic are in urgent need for daily-usage in medical environment, disease detection in resource limited area, and in- house infection diagnostics during global pandemics. There is a great need for multiplex detection of nucleic acids. Lateral flow assays are simple and highly successful analytical platform that can potentially fit those needs and has been used in various aspects such as infectious disease, toxins, pathogens. SUMMARY [0004] The technology described herein is directed to methods, composition, kits and systems for multiplex detection of target nucleic acids. In some embodiments of the various aspects described herein, low amounts of target nucleic acids, e.g., from multiple virus species are amplified simultaneously. The amplified product is processed to produce single stranded species which can be detected in a multiplex format to simultaneous detect two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) different target nucleic acids to achieve rapid, low cost, sensitive nucleic acid detection. Embodiments of the technology integrate one-pot multiplexed RPA (recombinase polymerization amplification), exonuclease digestions and multiplexed lateral flow assays. [0005] In one aspect, provided herein is a multiplex detection method for simultaneously detecting two or more different target nucleic acids. Generally, the method comprises preparing single-stranded or partially single-stranded amplicons from the target nucleic acids and detecting the single-stranded or partially single-stranded amplicons, for example, in a lateral flow assay or device. [0006] In some embodiments of any one of the aspects described herein, the method comprises amplifying the target nucleic acids to produce double-stranded amplicons and cleaving/digesting one strand of the double-stranded amplicons to produce the single-stranded amplicons. For example, the double-stranded amplicons can be treated with an exonuclease, e.g., 5’ to 3’ exonuclease to cleave/digest one strand of the double-stranded amplicons to produce single-stranded amplicons. For example, one strand of the double-stranded amplicons comprises a nucleic acid modification capable of inhibiting a 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease and the double-stranded amplicons are treated with a 5’ to 3’ exonuclease to produce single-stranded amplicons.

[0007] In some embodiments, one strand of the double-stranded amplicons comprises one or more uracil bases and the double-stranded amplicons are treated with a Uracil-DNA glycosylase (UDG) to produce single-stranded amplicons.

[0008] In some embodiments, the method comprises amplifying the target nucleic acids to produce double-stranded amplicons. One strand of the double-stranded amplicons comprises a nucleic acid modification capable of inhibiting a 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease. The double-stranded amplicons are treated with an exonuclease to digest one strand of the double-stranded amplicons to produce single-stranded amplicons. The presence of single-stranded amplicons can be detected, for example, in a lateral flow assay or device. [0009] In some embodiments of any of the aspects, the single-stranded amplicons are hybridized with a first nucleic acid probe and a second nucleic acid probe to form a complex prior to detection. Each first nucleic acid probe independently comprises a detection ligand and a nucleotide sequence substantially complementary to a first region of a single-stranded amplicon. The second nucleic acid probe comprises a nucleotide sequence substantially complementary to a second region of said single-stranded amplicon. Each second nucleic acid probe independently comprises a capture ligand so that the capture ligand in a first complex comprising a first single-stranded amplicon and first and second nucleic acid probes is distinguishable from a capture ligand in a second complex comprising a second single-stranded amplicon and first and second nucleic acid probes. The first and second single-stranded amplicons are different, e.g., have different nucleotide sequences.

[0010] The complexes comprising the single-stranded amplicon and first and second nucleic acid probes can be detected in a lateral flow assay configured to distinguish the capture ligand in a first complex from the capture ligand in a second complex. For example, the lateral flow assay can be configured for spatial multiplexed detection to distinguish the capture ligand in the first complex from the capture ligand in the second complex. In some embodiments of any one of the aspects, for example, the lateral flow assay can be carried out in lateral flow device, where the lateral flow device comprises: (i) a first test region comprising a first ligand binding molecule capable of binding with the capture ligand in the first complex comprising a first single-stranded amplicon and first and second nucleic acid probes; and (ii) a second test region comprising a second ligand binding molecule capable of binding with the capture ligand in the second complex comprising a second single-stranded amplicon and first and second nucleic acid probes.

[0011] In some embodiments of the various aspects, the ligand binding molecule capable of binding with the capture ligand is an antibody.

[0012] In some embodiments of any of the aspects, the single-stranded amplicons are hybridized with a first nucleic acid probe to form a complex prior to detection. Each first nucleic acid probe independently comprises a detection ligand and a nucleotide sequence substantially complementary to a first region of a single-stranded amplicon. One of the first nucleic acid probe comprises a nucleotide sequence substantially complementary to a first region of a first single-stranded amplicon and another of the first nucleic acid probe comprises a nucleotide sequence substantially complementary to a first region of a second single- stranded amplicon. The first and second single-stranded amplicons are different, e.g., have different nucleotide sequences.

[0013] The complexes comprising the single-stranded amplicon and the first nucleic acid probe can be detected in a lateral flow assay configured to distinguish the single-stranded amplicon in a first complex from the single-stranded amplicon in a second complex. For example, the lateral flow assay can be configured for spatial multiplexed detection to distinguish the single-stranded amplicon in the first complex from the single- stranded amplicon in the second complex. In some embodiments of any one of the aspects, the lateral flow assay can be carried out in a lateral flow device, where the lateral flow device comprises: (i) a first test region comprising a first nucleic acid capture probe comprising a nucleotide sequence substantially complementary to a second region of the single-stranded amplicon in a first complex; and (ii) a second test region comprising a second nucleic acid capture probe comprising a nucleotide sequence substantially complementary to a second region of the single-stranded amplicon in a second complex.

[0014] In some embodiments, the single-stranded amplicons comprise a detection ligand. The single-stranded amplicons comprising a detection ligand can be detected in a lateral flow assay configured to distinguish the different single-stranded amplicons. For example, the lateral flow assay can be configured for spatial multiplexed detection to distinguish the single- stranded amplicon based on their nucleotide sequences. In some embodiments of any one of the aspects, the lateral flow assay can be carried out in a lateral flow device, where the lateral flow device comprises: (i) a first test region comprising a first nucleic acid capture probe comprising a nucleotide sequence substantially complementary to a part of a first single- stranded amplicon; and (ii) a second test region comprising a second nucleic acid capture probe comprising a nucleotide sequence substantially complementary to a part of a second single- stranded amplicon.

[0015] In some embodiments, the single-stranded amplicons comprise an index domain. For example, the single-stranded amplicons comprise a sample identification/index domain, e.g., a domain/sequence for identifying the sample from which the amplified product came. The single-stranded amplicons comprising an index domain can be detected in a lateral flow assay configured to distinguish the different single-stranded amplicon. For example, the lateral flow assay can be configured for spatial multiplexed detection to distinguish the index domains of different single-stranded amplicons. In some embodiments of any one of the aspects, the lateral flow assay can be carried out in a lateral flow device, where the lateral flow device comprises: (i) a first test region comprising a first nucleic acid capture probe comprising a nucleotide sequence substantially complementary to at least part of the index domain of a first single-stranded amplicon; and (ii) a second test region comprising a second nucleic acid capture probe comprising a nucleotide sequence substantially complementary to at least part of the index domain of a second single-stranded amplicon.

[0016] In some embodiments, the method comprises amplifying a plurality of target nucleic acid to produce double-stranded amplicons. One strand of the double-stranded amplicon comprises at its 5’-end a detectable label and its 3’-end a sample identification/index domain, e.g., a domain/sequence for identifying the sample from which the amplified product came. After amplification, one strand, e.g., the strand lacking the detectable label at its 5’ -end, of the double-stranded amplicon is cleaved to produce single-stranded or at least partially single-stranded amplicons. The target nucleic acids from different samples can be amplified in one reaction vessel or in separate reaction vessels. If the amplification is in separate reactions vessels, the amplified product, the amplified product from at least two different samples can be pooled together. For example, the double-stranded amplicons from at least two different samples can be pooled together. Alternatively, or in addition, the single-stranded or at least partially single-stranded amplicons from at least two different samples can be pooled together. The single-stranded or at least partially single-stranded amplicons can be detected, for example, in a lateral flow assay or device. [0017] In some embodiments of any one of the aspects, the method comprises contacting the double-stranded amplicons with an exonuclease, e.g., an exonuclease having 5’ to 3’ cleaving activity. This produces single-stranded or at least partially single-stranded amplicons, which can be detected as described herein.

[0018] In some embodiments of any one of the aspects, the strand of double-stranded amplicon that lacks a detectable label at its 5’-end comprises at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) uridine nucleotide in a region complementary to the sample identification/index domain of the strand comprising a detectable label its 5’ -end, and the method comprises contacting the double-stranded amplicons with an Uracil-DNA glycosylase (UDG) to cleave the strand lacking the detectable label. This produces amplicons having a single-stranded region, e.g., a partially single-stranded amplicons, which can be detected as described herein.

[0019] In some embodiments, the single-stranded or partially single-stranded amplicons can be detected in a lateral flow assay configured to distinguish amplicons comprising different identification/index domains. For example, the lateral flow assay can be configured for spatial multiplexed detection to distinguish amplicons comprising different identification/index domains. For example, the lateral flow assay can be carried out in lateral flow device, where the lateral flow device comprises a plurality of capture/test regions. Each capture/test region independently comprises a nucleic acid probe immobilized thereon, wherein the nucleic acid probe comprises a nucleotide sequence substantially complementary to at least a part of an identification/index domain of a single-stranded or partially single-stranded amplicon, and wherein the immobilized nucleic acid probe of at least two capture/test regions are different. [0020] In some embodiments of any one of the aspects, the step of detecting comprises binding the detection ligand with a ligand binding molecule capable of binding with the detection ligand. Generally, the ligand binding molecule comprises a detectable label.

[0021] In some embodiments of the various aspects, the ligand binding molecule capable of binding with the detection ligand is an antibody.

[0022] The methods, compositions, kits and systems described herein can be used for multiplex detection of target nucleic acids. The target nucleic acids can be single-stranded or double-stranded. Further, the target nucleic acids can be DNA or RNA. In addition, the target nucleic acids can be viral or not be viral nucleic acids.

[0023] In another aspect, the disclosure provides a composition comprising one or more components and/or reagents described herein. [0024] In yet another aspect, the disclosure provides a kit comprising one or more components and/or reagents described herein.

[0025] In still another aspect, the disclosure provides a detection system comprising one or more components and/or reagents described herein.

BRIEF DESCRIPTION OF THE DRAWINGS [0026] FIGS. 1-5 are schematic representations of some exemplary embodiments of the multiplex detection method described herein.

[0027] FIGS. 6A-6E shows that combinatorial detection allows for even higher multiplexing.

[0028] FIGS. 7A and 7B shows methods of preparing multiplex lateral flow strips by placing different antibodies on different regions of the strip to achieve specific binding of target nucleic acid through antigen-antibody interactions (FIG. 7A) and by placing different nucleic acid probes (FIG. 7B) on different regions of the strip for target binding through hybridization. [0029] FIG. 8 shows some exemplary geometric arrangements of test regions for lateral flow strips.

[0030] FIG. 9A shows that screened SARS-CoV-2 primers can detect 10 copies of the virus RNA.

[0031] FIG. 9B is a schematic representation of the genomic location of the screened primers.

[0032] FIGS. 10A and 10B shows results for the screened human parainfluenza virus (HPIV) RPA primer via gel (FIG. 10A) and lateral flow detection (FIG. 10B).

[0033] FIG. 11 shows results of one-pot detection of HPIV and SARS-CoV-2 via gel (left panel) and lateral flow detection (right panel). Gel lanes from left to right in the left panel of FIG. 11: HPIV primer + HPIV RNA; HPIV primer; SARS-CoV-2 primer + SARS-CoV-2 RNA; SARS-CoV-2 primer; HPIV primer + SARS-CoV-2 primer + HPIV RNA + SARS-CoV- 2 RNA; HPIV primer + SARS-CoV-2 primer. In the right panel of FIG. 11, the printed LFD results can detect HPIV and SARS-CoV-2.

DETAILED DESCRIPTION

[0034] To achieve one-pot multiplexed RPA reaction, the inventors developed a computational algorithm that assists with the design of the primers to amplify their desired target region orthogonally. For the RPA reaction, the forward primer is modified with a 5' tail of 6 phosphorothioate-linked bases to confer exonuclease protection. After the multiplexed RPA reaction, the amplicon is subjected to the exonuclease digestion to produce single stranded DNA, exposing the binding site for the DNA probe to bind the target region, leading to the signal on the LFD devices. An exemplary embodiment of the method is shown in Figure 1. [0035] Figure 1 is a schematic of and exemplary multiplexed RPA and specific LFD of nucleic acid sequences (RNA or DNA) extracted from viruses or bacteria with multiplexed antigen printed strips. Step 1: Target viral RNA region (of domains denoted with a-b-c-d, e-f- g-h, and i-j-k-1 on different targets) is reverse transcribed into cDNA via extension of the reverse transcription primers by the Reverse Transcriptase in the reaction mixture. Subsequently, the cDNA is amplified via isothermal RPA at 42 °C by templated extension of the forward (a*, e*, i*) and reverse primers (d*, h*, 1*). The forward primer has a 6-nucleotide long poly-T segment with phosphorothioate bonds. Step 2a: Products of RPA are amended with SDS and transferred to the exo/LFD buffer that contains T7 exonuclease (a dsDNA- specific 5' to 3' exonuclease) and detection probes (one probe is labeled with a specific binding group (antigen), and another probe is labeled with FAM). The resulting mixture is incubated for 1 min at ambient temperature and the reverse strand of the dsDNA amplicon products get preferentially digested yielding ssDNA amplicon (a-b-c-d, e-f-g-h, i-j-k-1) homologous to the target RNA sequence. Step 2b: The 3' antigen modified (b*, f*, j*) and 5' FAM (c*, g*, k*) modified detection probes will allow the desired target to bind to the printed strips. The strip is printed with different antibodies to be visualized (e.g., antibodies specific to target proteins or tags; see e.g., Fig. 7A). The right ssDNA amplicon acts as a bridge that binds both the antigen-probe and the FAM-probe, then the gold nanoparticle labeled with FAM-antibody will accumulate on the target line to produce a colored line to show positive signal. The control line formed of rabbit secondary antibodies captures the remaining gold nanoparticle conjugates by binding to rabbit anti-FAM IgG.

[0036] Figure 2 is a schematic of another exemplary multiplexed RPA and specific lateral flow detection of nucleic acid sequences (RNA or DNA) extracted from viruses or bacteria with multiplexed DNA probe printed strips. Step 1: Target viral RNA region (of domains denoted with a-b-c-d, e-f-g-h, and i-j-k-1 on different targets) is reverse transcribed into cDNA via extension of the reverse transcription primers by the Reverse Transcriptase in the reaction mixture. Subsequently, the cDNA is amplified via isothermal RPA at 42 °C by templated extension of the forward (a*, e*, i*) and reverse primers (d*, h*, 1*). The forward primer has a 6-nucleotide long poly-T segment with phosphorothioate bonds. Step 2a: Products of RPA are amended with SDS and transferred to the exo/LFD buffer that contains T7 exonuclease (a dsDNA-specific 5' to 3' exonuclease) and detection probes (one probe is labeled with a specific binding group (antigen), and another probe is labeled with FAM). The resulting mixture is incubated for 1 min at ambient temperature and the reverse strand of the dsDNA amplicon products get preferentially digested yielding ssDNA amplicon (a-b-c-d, e-f-g-h, i-j-k-1) homologous to the target RNA sequence. Step 2b: 5' FAM (c*, g*, k*) modified detection probes will allow the desired target to bind to the printed strips. The strip is printed with multiplexed binding probes (b*, f*, j*) to different targets. The right ssDNA amplicon acts as a bridge that binds both the probe on the strip and the FAM-probe, then the gold nanoparticle labeled with FAM-antibody will accumulate on the target line to produce a colored line to show positive signal. The control line formed of rabbit secondary antibodies captures the remaining gold nanoparticle conjugates by binding to rabbit anti-FAM IgG.

[0037] Embodiments of the technology described herein, mtsRPA (multiplex toehold switching-based RPA FIGS. 3-5), take advantage of high sequence specificity of toehold switching to achieve high multiplexing and high accuracy. It utilizes amplification, e.g., rapid isothermal Recombinase Polymerase Amplification (RPA), nanoparticles, and Lateral Flow Detection (LFD) for ultrafast amplification and detection.

[0038] In an exemplary embodiment for detecting viral RNA as an example, the target nucleic acid, e.g., viral RNA can first be extracted or enzymatically released. Each prepared sample can have a designated barcode/index pij. For each sample, the viral genome containing the target sequence x can be added into a reaction mix with reverse transcriptase, RPA reagents, Forward primer a labeled with nanoparticles or fluorophore, and reverse primer b with sample index pij. a binds to the complementary sequence a* in target and reverse transcription converts the RNA into RNA-cDNA hybrid. Recombinase then allows for reverse primer b- p,. _j to bind to b* sequence on the cDNA and generate a full amplicon of a*-x-b- pij. RPA then exponentially amplifies the amplicon and generates a detectable signal in 5 minutes. The samples can be pooled after amplification. The pooled product can be added to an LFD pad where index sequence p can be used for toehold switch. On the LFD pad, there are multiple spots with different index sequences. The binding of nanoparticle/fluorophore-bound strand to the probes on the LFD pad in a spot specific manner allows for that designated spot to show up on the device for signal verification. The barcode sequence can be designed such that even a one base mismatch between the intended target sequence and the actual sequence inhibits the capture efficiency by many orders of magnitude, ensuring specificity of detection. The capture of the amplicon concentrates the fluorescent or nanoparticle tag and causes a visible color change at that location, positively identifying the presence of the molecular target. Exemplary embodiments are shown in FIGS. 3 and 4. [0039] Figure 3 is a schematic of an exemplary embodiment. In this embodiment, after amplification with RPA, the full amplicon is treated with an exonuclease, e.g., Lambda exonuclease or T7 exonuclease to remove the a*-x-b- p _ij strand. Since the a-x*-b*- p* _ij trand is bound to nanoparticles or fluorophores, it is protected from digestion with the exonuclease. The samples can be pooled and loaded on an LFD pad where different capture zones are printed with the double-stranded probe with p,., overhang. The p* _ij on the target strand binds to the p*ij overhang in the probe and displaces the a-x*-b* probe strand due to longer base matching. The signal from the nanoparticles or fluorophore is then enriched on LFD. Based on the spots that have signals, positive samples can be identified.

[0040] Figure 4 is a schematic of an exemplary embodiment. In this embodiment, the reverse transcription primers comprise one or more uracil bases in the identification/index domain/sequence. After amplification, the amplified product can be contacted with an Uracil- DNA glycosylase to fragment the index sequence p* _ij in the reverse primers and expose the complementary sequence p* _ij in the full amplicons. The digested samples can then be pooled added to an LFD pad. The single-stranded p* _ij binds to the p* _ij sequence in the probes and the complementary strand ( a*-x-b ) in the full amplicon will be displaced by the probes due to longer base matching. The signal from the nanoparticles or fluorophore is then enriched on LFD. Based on the spots that have signals, positive samples can be identified.

[0041] Figure 5 is a schematic of another exemplary embodiment. In this embodiment, phosphorothioate bases are incorporated in one of the primers, e.g., the forward primers. After RPA, the full amplicon can be treated with an exonuclease, e.g., Lambda exonuclease or T7 exonuclease to remove the a*-x-b- p* _ij strand. The a-x*-b*~ p* _ij strand will be protected by the phosphorothioate internucleotide linages. The samples can be pooled and loaded on an LFD pad where only the p* _ij sequence is printed on the pad. A nucleic acid probe with a detectable label can be added for detecting the captured a-x*-b*~ p* _ij strand.

[0042] Combinatorial barcoding of samples followed by subsequent detection on a lateral flow device: DNA/RNA samples (like genetic material from viruses, bacteria, animals, plants etc. and also synthetic nucleic acid molecules) may be combinatorially barcoded during amplification with a set of primers carrying different barcodes. In this case, during amplification, different amplicons of the target will carry distinct barcodes. When these amplicons are converted into single stranded form (with exonuclease digestion) or a portion of a single strand is exposed (by USER enzyme digestion) they reveal the barcoded sequence regions which can then bind to the corresponding positions on the LFD, as described previously. Multiple positions on the LFD, corresponding to the particular sample, will show signal allowing us to combinatorially encode a potential 2 ^Λ h distinct samples into n spots on an LFD. See, FIGS. 6A-6E.

[0043] One challenge for the multiplexed detection is the readout on the LFD devices. To solve this problem, two different methods for the LFD strips were developed. As shown in Figures 7A and 7B, one way to address this challenge is to conjugate different antibodies on different test regions of a lateral flow strip (Figure 7A). Another way to is to conjugate different nucleic acid capture probes on different test regions to allow multiplexed sequence specific binding of the target. For example, biotin labeled DNA can be conjugated to different lane of streptavidin.

[0044] It is noted that the test regions in a lateral flow strip for multiplexing do not have to be arranged in linear pattern. Other geometric patterns could also be used. Some exemplary different geometric LFD devices that can achieve spatial multiplexed detection are shown in FIG. 8. The target pads can be patterned with antibodies and/or capture probes as described in FIGS. 1-5

[0045] In Figure 9A, a mixture of 2.5 μl each of 10 μM forward and reverse primers to the specified target, 29.5 μl of TWISTAMP BASIC RPA rehydration buffer (TWISTDX, TABAS03KIT), 5 μl of DNase/RNase-free water, and 0.5 μl of PROTOSCRIPT II reverse transcriptase (NEB, M0368S) were vortexed briefly and added to the TWISTAMP lyophilized powder mix. Then, 5 μl of sample was added to the tube while 5 μl of 280 mM Magnesium Acetate was added to the reaction tube lid. The 50 μl mixture was vortexed briefly, spun down, vortexed again, spun down once more, and immediately incubated at 42 °C for 5 min in a standard PCR machine (APPLIED BIOSYSTEMS, 4484073) or heating block (BENCHMARK SCIENTIFIC, BSH300). During amplification, a digestion and LFD buffer mixture was prepared with 64 μl of LFD running buffer (MILENIA, MGHD 1), 1 μl of 10 μM biotin probes, 1 μl of 10 μM FAM probes, 10 μl of 10x NEBUFFER 4 (NEB, B7004S), and 4 μl of T7 exonuclease (NEB, M0263S), in a total volume of 80 μl. The mixture was vortexed briefly and added to a 2 mL lo-bind tube (EPPENDORF). Once complete, 8 μl of the RT-RPA reaction was subsequently mixed thoroughly and completely with a 10% Sodium Dodecyl Sulfate (SDS) at a ratio of 8 μl sample: 12 μl SDS to inactivate enzymes. This 20 μl mixture was then added to the 80 μl digestion mixture above and incubated for 1 min at room temperature.

[0046] In Figure 10A, 4 set of primers were mixed with HPIV RNA template with TWIST RPA kit and Reverse transcriptase. The recipe was same as the Figure 9A. After the amplification reaction, 1 uL of the products were used for the gel electrophoresis. All gels were denaturing PAGE at 15% polyacrylamide (INVITROGEN, EC6885BOX), run in lx TBE buffer that was diluted from 10x TBE (PROMEGA, V4251) with filtered water, at RT °C, 200V, for 30 min. Immediately after 5 min of RT-RPA, 5 μl of products were directly mixed with 5 μl of formamide dye consisting of 100% formamide (THERMOFISHER) and Bromophenol blue dye (SIGMA ALDRICH). Gels were then removed from cassettes, stained in 1x SYBRSAFE (LIFE TECHNOLOGIES) for 3 min, and imaged with a TYPHOON scanner (GENERAL ELECTRIC).

[0047] In Figure 10B, after the amplification, a digestion and LFD buffer mixture was prepared with 64 μl of LFD running buffer (MILENIA, MGHD 1), 1 μl of 10 μM biotin probes, 1 μl of 10 μM FAM probes, 10 μl of 10x NEBUFFER 4 (NEB, B7004S), and 4 μl of T7 exonuclease (NEB, M0263S), in a total volume of 80 μl. The mixture was vortexed briefly and added to a 2 mL lo-bind tube (EPPENDORF). Once complete, 8 μl of the RT-RPA reaction was subsequently mixed thoroughly and completely with a 10% Sodium Dodecyl Sulfate (SDS) at a ratio of 8 μl sample: 12 μl SDS to inactivate enzymes. This 20 μl mixture was then added to the 80 μl digestion mixture above and incubated for 1 min at room temperature. [0048] In Figure 11, both the COVID and HPIV RPA primer were mixed together for the RPA amplification according to the same protocol in Figure 9 and Figure 10. After the amplification, 1 uL of the product were used for the gel running in the left panel of Figure 11. For the right panel of Figure 11, both the DNA probe for HPIV and SARS-CoV-2 was printed on the LFD substrate. The LFD was inserted into a buffer containing the beads modified with capture probe for amplification products. The RPA amplification products were added to the LFD, and the reaction was run for 10 mins.

Nucleic acid modifications

[0049] In some embodiments of any of the aspects, single-stranded amplicons or one of the primers used for amplifying the target nucleic acids comprises a nucleic acid modification capable of inhibiting 5 ’ to 3 ’ cleaving activity of a 5 ’ to 3 ’ exonuclease. In some embodiments of any of the aspects, the forward primer comprises a nucleic acid modification capable of inhibiting 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease. It is noted that either the forward or the backward/reverse primer can comprise the nucleic acid modification capable of inhibiting 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease. Accordingly, in some embodiments of any of the aspects, the forward primer comprises a nucleic acid modification capable of inhibiting 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease. In some other embodiments of any of the aspects, the backward/reverse primer comprises a nucleic acid modification capable of inhibiting 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease. OOOO0] In some embodiments, the primer comprising a modification capable of inhibiting 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease also comprises a detection ligand. Similarly, in some embodiments, the single-stranded amplicon comprising a modification capable of inhibiting 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease also comprises a detection ligand. [0051] The modification capable of inhibiting 5’ to 3’ cleaving activity can be present anywhere in the single-stranded amplicon or the primer. For example, it can be at the 5’ -end or terminus, at an internal position, or at a position within the 5’ -terminus, e.g., at positions within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 bases from the 5’-end of the single- stranded amplicon or the primer. In some embodiments of any of the aspects, the nucleic acid modification is located at the 5’ -end of the single-stranded amplicon or the primer.

[0052] Nucleic acid modifications that can inhibit 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease are known in the art, such as modified internucleotide linkages, modified nucleobase, modified sugar, and any combinations thereof. Exemplary modifications include, but are not limited to 1, 2, 3, 4, 5, 6 or more modified internucleotide linkages, such as phosphorothioates; an inverted nucleoside or 5’ to 5’ intemucleotide linkage; a 3’ to 3’ internucleotide linkage; a 2’-OH or a 2’-modified nucleoside; a 5’-modified nucleotide; a 2' to 5’ linkage; an abasic nucleoside; an acyclic nucleoside; replacement of 5’ -OH group; a spacer; left-handed DNA; nucleotides with non-canonical nucleobases; or any combinations thereof. [0053] Exemplary modified intemucleotide linkages include, but are not limited to, phosphorothioates, phosphorodithioates, phosphotriesters, alkylphosphonates, phosphoramidate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, alkyl or aryl phosphonates, bridged phosphoroamidates, bridged phosphorothioates, bridged alkylenephosphonates, methylenemethylimino ( — CH ₂ -N(CH3)- O — CH ₂ -), thiodiester ( — O — C(O) — S — ), thionocarbamate ( — O — C(O)(NH) — S — ), siloxane ( — O — Si(H)2-0 — ), and N,N'-dimethylhydrazine ( — CH ₂ -N(CH3)-N(CH3)-), amide- 3 (3'- CH ₂ -C(=O)-N(H)-5'), amide-4 (3'-CH ₂ -N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH ₂ -0-5'), formacetal (3 O —CH ₂ -0-5'), oxime, methyleneimino, methykenecarbonylamino, methylenemethylimino (MMI, 3'-CH ₂ -N(CH3)-0-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’ O O — C5’), thioethers (C3’-S-C5’), thioacetamido (C3’-N(H)-C(=O)-CH ₂ -S-C5\ C3’-0-P(O)-0- SS-C5’, C3 ’ -CH ₂ -NH-NH-C 5 ’ , 3'-NHP(O)(0CH ₃ )-0-5' and 3'-NHP(O)(0CH3)-0-5\ In some embodiments of any of the aspects, the modified intemucleotide linkages are phosphorothioate. [0054] Exemplary 2' modifications include, but are not limited to, 2' -halo ( e.g ., 2’-fluoro), 2’-alkoxy (e.g., 2’-0-methyl and 2’-0-methylmethoxy), 2’-aryloxy, 2’-0-amine or 2’-0- alkylamine (amine NFL·; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, dihet.eroaryl amino, ethylene diamine or polyamino), O- CH ₂ CH ₂ (NCH ₂ CH ₂ NMe2)2, methyleneoxy (4'-CH ₂ -0-2') LNA, ethyleneoxy (4'-(CH ₂ )2-0-2') ENA, 2’-amino (e.g. 2’ -NIL·, 2’ -alkylamino, 2’-dialkylamino, 2’-heterocyclylamino, 2' - arylamino, 2’-diaryl amino, 2’-heteroaryl amino, 2' -diheteroaryl amino, and 2’-amino acid); NF[(CFbCFbNF[)nCFbCFb-AMINE (AMINE = NH ₂ , alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino), -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), 2’-cyano, 2’-mercapto, 2’-alkyl-thio-alkyl, 2' - thioalkoxy, 2’-thioalkyl, 2’-alkyl, 2' -cycloalkyl, 2’-aryl, 2’-alkenyl and 2’-alkynyl.

[0055] Exemplary modification for 5’-OH group include, but are not limited to 5'- monothiophosphate (phosphorothioate), 5'-monodithiophosphate (phosphorodithioate), 5'- phosphorothiolate, 5'-alpha-thiotriphosphate, 5’-beta-thiotriphosphate, 5'-gamma- thiotriphosphate, 5'-phosphoramidates, 5'-alkylphosphonate, 5'-alkyletherphosphonate, a detection ligand, a detectable label, a capture ligand, a tag molecule and a ligand. Non-limiting examples of detection ligands, detectable labels and capture ligands are described further herein.

[0056] Exemplary inverted nucleosides include dT. For example, a dT linked via its 5’- end to the 5’ -end of nucleoside in the single-stranded amplicon or the primer. Inverted nucleosides also include nucleosides linked by a 2' to 2’, 2' to 3’, 2' to 2' or 3’ to 3’ internucleotide linkage.

[0057] Non-limiting examples of spacers for use as modifications for inhibiting 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease include, but are not limited to, the C3 propyl spacer (propanediol), hexanediol, l’^’-Dideoxyribose (dSpacer), PC (Photo-Cleavable) Spacer, Spacer 9 (a tri ethylene glycol spacer), and Spacer 18 (an 18-atom hexa-ethyleneglycol spacer). [0058] In some embodiments of any of the aspects, the left-handed DNA is Z-DNA. Z- DNA is one of the possible double helical structures of DNA. It is a left-handed double helical structure in which the helix winds to the left in a zigzag pattern, instead of to the right, like the more common B-DNA form. Z-DNA is one of three biologically active double-helical structures along with A- and B-DNA. Many enzymes (e.g., exonucleases) that use right-handed DNA as a substrate cannot use left-handed DNA as substrate

[0059] In some embodiments of any of the aspects, the single-stranded amplicon or the primer with the modification capable of inhibiting the 5’ to 3’ cleaving activity comprises 1, 2, 3, 4, 5, 6 or more modified internucleotide linkages, e.g. phosphorothioate linkages. It is noted that when the single-stranded amplicon or the primer comprises two or more modified internucleotide linkages, they can be present next to each other, i.e., at consecutive positions or they could comprise unmodified intemucleotide linkages, i.e., phosphodiesters at 1 or more nucleotides between two modified intemucleotide linkages. Preferably, when the single- stranded amplicon or the primer comprises two or more modified intemucleotide linkages, the modified intemucleotide linkages are next to each other, i.e., at consecutive positions.

[0060] In some embodiments of any of the aspects, the single-stranded amplicon or the primer comprising the modification capable of inhibiting the 5’ to 3’ cleaving activity comprises a poly(dT) sequence at the 5’-terminus. For example, the single-stranded amplicon or the primer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more dT nucleotides at the 5’-terminus. [0061] In some embodiments of any of the aspects, the single-stranded amplicon or the primer comprising the modification capable of inhibiting the 5’ to 3’ cleaving activity comprises a poly(dT) sequence at the 5’-terminus and 1, 2, 3, 4, 5, 6 or more modified intemucleotide linkages, e.g., phosphorothioate. In some embodiments of any of the aspects, the modified intemucleotide linkages are in the poly(dT) sequence. For example, the modified intemucleotide linkages are present starting at position 1 from the 5’ -end.

[0062] In some embodiments, the single-stranded amplicon or the primer comprising the modification capable of inhibiting the 5’ to 3’ cleaving activity comprises a detection ligand at the 5’ -end.

[0063] In some embodiments, the single-stranded amplicon or the primer comprising the modification capable of inhibiting the 5’ to 3’ cleaving activity comprises a capture ligand at the 5’ -end.

[0064] It is noted that a single-stranded amplicon or primer comprising the modification capable of inhibiting the 5’ to 3’ cleaving activity can comprise one or more of the modifications described herein. For example, the single-stranded amplicon or primer can comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modifications independently selected from the group consisting of: (i) 1, 2, 3, 4, 5, 6 or more modified intemucleotide linkages; (ii) an inverted nucleoside or 5’ to 5’ intemucleotide linkage; (iii) a 2’-OH or a 2' - modified nucleoside; (iv) a 5’ -modified nucleotide; (v) a 2’ to 5’ linkage; (vi) an abasic nucleoside; (vii) an acyclic nucleoside; (viii) a spacer; (ix) left-handed DNA; (x) non-canonical nucleobases nucleotide; and (xii) any combinations of (i)-(x).

[0065] Generally, the primer lacking the modification for inhibiting the 5’ to 3’ cleavage activity comprises a 5’ -OH or nucleic acid modification capable of enhancing the 5’ to 3’ cleaving activity of an exonuclease, for example, a phosphate group at the 5’ -end. Accordingly, in some embodiments of any of the aspects, the primer lacking the modification for inhibiting the 5’ to 3’ cleavage activity comprises a 5’-OH or a 5’-phosphate group, e.g., a 5'-monophosphate; 5'-diphosphate or a 5'-triphosphate at the 5’ -end. In some embodiments of any of the aspects, one or both of the first or second primers comprises a phosphate group at the 5’ -end.

[0066] In some embodiments, a primer lacking the modification for inhibiting the 5’ to 3’ cleavage activity comprises a 5’-OH or a nucleic acid modification capable of enhancing the 5’ to 3’ cleaving activity of an exonuclease. For example, the primer comprises a phosphate group at the 5’ -end.

[0067] In some embodiments, one of the forward or backward/reverse primer comprises a modification capable of inhibiting the 5’ to 3’ cleavage activity and the other of the forward or backward/reverse primer comprises a 5’ -OH or a nucleic acid modification capable of enhancing the 5’ to 3’ cleaving activity of an exonuclease. Use of such a pair of primers produces double-stranded amplicons where one strand is protected against cleavage while the other strand is preferentially cleaved.

Index/barcode domain

[0068] In some embodiments of any one of the aspects described herein, the single- stranded amplicon comprises an index or barcode domain. As used herein, an “index” or “barcode” domain is a sequence of nucleotides that uniquely identifies a particular molecule. Barcode domain may also be referred to in the art as “unique molecular identifiers” (UMIs). UMIs associate distinct sequences with a nucleic acid molecule and can be used to uniquely identify an amplified nucleic acid molecule or the sample from which came the amplified nucleic acid molecule. In some embodiments, the barcode domain may contain a nucleotide sequence that contains only three of the four nucleotides. For example, a barcode domain or subdomain may include (a) only As, Ts, and Cs, (b) only As, Ts, and Gs, (c) only Gs, Ts, and Cs, or (d) only As, Gs, and Cs, Thus, a barcode domain or subdomain may lack (may not include) one of A, T, C or G. The length of a barcode domain may also vary as desired.

[0069] It is noted that the index domain can be present anywhere in the single-stranded amplicon. For example, it can be at the 3’ -end or terminus, at an internal position, or at a position within the 3’-terminus, e.g., within positions within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 from the 3’ -end of the single-stranded amplicon. In some embodiments of any of the aspects, the index domain is located at the 3’ -end of the single-stranded amplicon. [0070] In some embodiments of any one of the aspects described herein, one stand of the double-stranded amplicon comprises an index domain. In some further embodiments, the complementary strand comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more uracil bases in a region complementary to the index domain.

Amplification

[0071] Embodiments of the various aspects described herein comprise a step of amplifying the target nucleic acid. As used herein, “amplification” is defined as the production of additional copies of a nucleic acid sequence, i.e., for example, amplicons or amplification products. Methods of amplifying nucleic acid sequences are well known in the art. Such methods include, but are not limited to, isothermal amplification, polymerase chain reaction (PCR) and variants of PCR such as Rapid amplification of cDNA ends (RACE), ligase chain reaction (LCR), multiplex RT-PCR, immuno-PCR, SSIPA, Real Time RT-qPCR and nanofluidic digital PCR. Accordingly, the methods described herein comprise a step of contacting a target nucleic acid with a DNA polymerase and a set of primers. In some embodiments of any of the aspects, a set of primers comprises at least 2 primers and comprises a forward primer and reverse primer that amplify a target of about 50 base pairs (bp) to about 50,000 bp, unless indicated otherwise.

[0072] It is noted that the target nucleic acids can be amplified separately or together. For example, the target nucleic acids can be amplified separately and the produced double-stranded amplicons pooled together prior to producing the single-stranded amplicons. Alternatively, or in addition, the target nucleic acids can be pooled together prior to amplification [0073] Methods for amplifying nucleic acids are well known in the art and amenable to the methods, composition, kits, and systems described herein. In some embodiments of any of the aspects, the amplification step comprises isothermal amplification. As used herein, “isothermal amplification” refers to amplification that occurs at a single temperature. For example, the amplification process is performed at a single temperature or where the major aspect of the amplification process is performed at a single temperature. Generally, isothermal amplification relies on the ability of a polymerase to copy the template strand being amplified to form a bound duplex. Isothermal amplification permits rapid and specific amplification of a target nucleic acid at a constant temperature. In general, isothermal amplification is comprised of (i) sequence-specific hybridization of primers to sequences within a target nucleic acid, and (ii) subsequent amplification involving multiple rounds of primer annealing, elongation, and strand displacement (as a non-limiting example, using a combination of recombinase, single-stranded binding proteins, and DNA polymerase). The primers used in isothermal amplification are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to a strand of the target nucleic acid to be amplified.

[0074] Non-limiting examples of isothermal amplification include: Recombinase Polymerase Amplification (RPA), Loop Mediated Isothermal Amplification (LAMP), Helicase-dependent isothermal DNA amplification (HDA), Rolling Circle Amplification (RCA), Nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR), and polymerase Spiral Reaction (PSR). See e.g., Yan et al., Isothermal amplified detection of DNA and RNA, March 2014, Molecular BioSystems 10(5), DOL 10.1039/c3mb70304e, the content of which is incorporated herein by reference in its entirety.

[0075] In some embodiments of any of the aspects, the isothermal amplification reaction(s) is Recombinase Polymerase Amplification (RPA), i.e., the step of amplifying the target nucleic acids comprises Recombinase Polymerase Amplification. RPA is a low temperature DNA and RNA amplification technique. The RPA process employs three core enzymes - a recombinase, a single-stranded DNA-binding protein (SSB) and strand-displacing polymerase. Recombinases are capable of pairing oligonucleotide primers with homologous sequence in duplex DNA. SSB bind to displaced strands of DNA and prevent the primers from being displaced. Finally, the strand displacing polymerase begins DNA synthesis where the primer has bound to the target DNA. By using two opposing primers, much like PCR, if the target sequence is indeed present, an exponential DNA amplification reaction is initiated. No other sample manipulation such as thermal or chemical melting is required to initiate amplification. At optimal temperatures (e.g., 37-42 °C), the RPA reaction progresses rapidly and results in specific DNA amplification from just a few target copies to detectable levels, typically within 10 minutes, for rapid detection of the target nucleic acid. In some embodiments of any of the aspects, the single-stranded DNA-binding protein is a gp32 SSB protein. In some embodiments of any of the aspects, the recombinase is a uvsX recombinase. See e.g., US Patent 7,666,598, the content of which is incorporated herein by reference in its entirety. In some embodiments of any of the aspects, RPA can also be referred to as Recombinase Aided Amplification (RAA). Accordingly, in some embodiments of any of the aspects, the amplification step comprises contacting the sample with a recombinase and single-stranded DNA binding protein. In some embodiments of any of the aspects, the amplification step comprises contacting the sample with a strand-displacing DNA polymerase, a set of primers, a recombinase, and single-stranded DNA binding protein.

[0076] In some embodiments of any of the aspects, the isothermal amplification reaction(s) is Loop Mediated Isothermal Amplification (LAMP), i.e., the step of amplifying the target nucleic acids comprises Loop Mediated Isothermal Amplification. LAMP is a single tube technique for the amplification of DNA; LAMP uses 4-6 primers, which form loop structures to facilitate subsequent rounds of amplification. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample with a strand-displacing DNA polymerase and a set of primers, wherein the set of primers comprises 4, 5, or 6 loop-forming primers.

[0077] In some embodiments of any of the aspects, the isothermal amplification reaction(s) is Helicase-dependent isothermal DNA amplification (HDA). HDA uses the double-stranded DNA unwinding activity of a helicase to separate strands for in vitro DNA amplification at constant temperature. In some embodiments of any of the aspects, the helicase is a thermostable helicase, which can improve the specificity and performance of HDA; as such, the isothermal amplification reaction(s) can be thermophilic helicase-dependent amplification (tHDA). As a non-limiting example, the helicase is the thermostable UvrD helicase (Tte-UvrD), which is stable and active from 45°C to 65 °C. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample with a DNA polymerase, a set of primers, and a helicase, wherein the helicase is optionally a thermostable helicase.

[0078] In some embodiments of any of the aspects, the isothermal amplification reaction(s) is Rolling Circle Amplification (RCA). RCA starts from a circular DNA template and a short DNA or RNA primer to form a long single stranded molecule. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample (e.g., a circular DNA) with a DNA polymerase and a set of primers, wherein the second set of primers comprises a single primer.

[0079] In some embodiments of any of the aspects, the isothermal amplification reaction(s) is Nucleic acid sequence-based amplification (NASBA), which is also known as transcription mediated amplification (TMA). NASBA is an isothermal technique predominantly used for the amplification of RNA through the cyclic formation of complimentary DNA and destruction of original RNA sequence (e.g., using RNase H). The NASBA reaction mixture contains three enzymes — reverse transcriptase (RT), RNase H, and T7 RNA polymerase — and two primers. T7 RNA Polymerase is an RNA polymerase from the T7 bacteriophage that catalyzes the formation of RNA from DNA in the 5'® 3' direction. Primer 1 (PI) contains a 3' terminal sequence that is complementary to a sequence on the target nucleic acid and a 5' terminal (+)sense sequence of a promoter that is recognized by the T7 RNA polymerase. Primer 2 (P2) contains a sequence complementary to the P1 -primed DNA strand. The NASBA enzymes and primers operate in concert to amplify a specific nucleic acid sequence exponentially. NASBA results in the amplification of the target RNA to cDNA to RNA to cDNA, etc., with alternating reverse transcription (e.g., RNA to DNA) and transcription steps (e.g., DNA to RNA), and the RNA being degraded after each transcription. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample (e.g., a cDNA) with an RNA polymerase, a reverse transcriptase, RNaseH, and a set of primers, wherein the set of primers comprise a 5’ sequence that is recognized by the RNA polymerase.

[0080] In some embodiments of any of the aspects, the isothermal amplification reaction(s) is Strand Displacement Amplification (SDA). SDA is an isothermal, in vitro nucleic acid amplification technique based upon the ability of the restriction endonuclease HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo-klenow) DNA polymerase to extend the 3 '-end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as target for an antisense reaction and vice versa. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample with a DNA polymerase (e.g., exo-klenow), a set of primers, and a restriction endonuclease (e.g., HincII).

[0081] In some embodiments of any of the aspects, the isothermal amplification reaction(s) is nicking enzyme amplification reaction (NEAR), which is a similar approach to SDA. In NEAR, DNA is amplified at a constant temperature (e.g., 55 °C to 59 °C) using a polymerase and nicking enzyme. The nicking site is regenerated with each polymerase displacement step, resulting in exponential amplification. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample with a DNA polymerase (e.g., exo-klenow), a set of primers, and a nicking enzyme (e.g., N.BstNBI).

[0082] In some embodiments of any of the aspects, the isothermal amplification reaction(s) is Polymerase Spiral Reaction (PSR). The PSR method employs a DNA polymerase (e.g., Bst) and a pair of primers. The forward and reverse primer sequences are reverse to each other at their 5’ end, whereas their 3’ end sequences are complementary to their respective target nucleic acid sequences. The PSR method is performed at a constant temperature 61 °C- 65 °C, yielding a complicated spiral structure. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample with a DNA polymerase (e.g., exo-klenow) and a set of primers that are reverse to each other at their 5’ end.

[0083] In some embodiments of any of the aspects, the isothermal amplification reaction(s) is polymerase cross-linking spiral reaction (PCLSR). PCLSR uses three primers (e.g., two outer-spiral primers and a cross-linking primer) to produce three independent prerequisite spiral products, which can be cross-linked into a final spiral amplification product. Accordingly, in some embodiments of the aspects, the amplification step comprises contacting the sample with a DNA polymerase and a set of primers (e.g., two outer-spiral primers and a cross-linking primer).

[0084] In some embodiments of any of the aspects, the DNA polymerase used in the amplification step is a strand-displacing polymerase. The term strand displacement describes the ability to displace downstream DNA encountered during synthesis. In some embodiments of any of the aspects, at least one (e.g. 1, 2, 3, or 4) strand-displacing DNA polymerase is selected from the group consisting of: Polymerase I Klenow fragment, Bst polymerase, Phi-29 polymerase, and Bacillus subtilis Pol I (Bsu) polymerase. In some embodiments of any of the aspects, step (c) comprises contacting the sample (e.g., cDNA) with the strand-displacing DNA polymerases Polymerase I Klenow fragment, Bst polymerase, Phi-29 polymerase, and Bacillus subtilis Pol I (Bsu) polymerase.

[0085] In some embodiments of any of the aspects, the DNA polymerase is provided (i.e., added to the reaction mixture) at a sufficient concentration to promote polymerization, e.g., 0.1 U/μL to 100 U/μL. As used herein, one unit (“U”) of DNA polymerase is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 37°C.

[0086] In some embodiments of any of the aspects, the sample is contacted with at least one set of primers. In some embodiments of any of the aspects, the set of primers is specific to the target nucleic acid. In some embodiments of any of the aspects, the set of primers is specific (i.e., binds specifically through complementarity) to cDNA. In other words, the DNA produced in the RT step that is complementary to a target RNA.

[0087] In some embodiments of any of the aspects, the isothermal amplification step, e.g., RPA is performed at a temperature between from about 12°C to about 45°C. As a non-limiting example, the isothermal amplification step is performed at least 12°C, at least 13°C, at least 14°C, at least 15°C, at least 16°C, at least 17°C, at least 18°C, at least 19°C, at least 20°C, at least 21°C, at least 22°C, at least 23°C, at least 24°C, at least 25°C, at least 26°C, at least 27°C, at least 28°C, at least 29°C, at least 30°C, at least 31°C, at least 32°C, at least 33°C, at least 34°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 39°C, at least 40°C, at least 41°C, at least 42°C, at least 43°C, at least 44°C, or at least 45°C.

[0088] In some embodiments of any of the aspects, the isothermal amplification step, e.g., RPA is performed at a temperature of at most 12°C, at most 13°C, at most 14°C, at most 15°C, at most 16°C, at most 17°C, at most 18°C, at most 19°C, at most 20°C, at most 21°C, at most 22°C, at most 23°C, at most 24°C, at most 25°C, at most 26°C, at most 27°C, at most 28°C, at most 29°C, at most 30°C, at most 31°C, at most 32°C, at most 33°C, at most 34°C, at most 35°C, at most 36°C, at most 37°C, at most 38°C, at most 39°C, at most 40°C, at most 41°C, at most 42°C, at most 43°C, at most 44°C, or at most 45°C.

[0089] In some embodiments of any of the aspects, the isothermal amplification step, e.g., RPA is performed at a temperature of about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23 °C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, or about 45°C. [0090] In some embodiments of any of the aspects, the isothermal amplification step, e.g., RPA is performed at room temperature (e.g., 20°C-22°C). In some embodiments of any of the aspects, the isothermal amplification step is performed at body temperature (e.g., 37°C). In some embodiments of any of the aspects, the isothermal amplification step, e.g., RPA is performed at about 42°C, e.g., on a heat block set to approximately 42°C.

[0091] The amplification step such as isothermal amplification step, e.g., RPA can be performed for any period of time to produce the double-stranded amplicons. For example, the amplification step, RPA can be for a period of from about 5 minutes to about 4 hours.

[0092] In some embodiments of any of the aspects, the amplification step, e.g., isothermal amplification step, e.g., RPA, is performed for about 5 minutes. As a non-limiting example, the isothermal amplification step is performed for about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 60 minutes, about 70 minutes, about 75 minutes, about 80 minutes, or about 90 minutes.

[0093] In some embodiments of any of the aspects, the amplification step, e.g., isothermal amplification step, e.g., RPA is performed for at most 5 minutes, at most 6 minutes, at most 7 minutes, at most 8 minutes, at most 9 minutes, at most 10 minutes, at most 11 minutes, at most 12 minutes, at most 13 minutes, at most 14 minutes, at most 15 minutes, at most 16 minutes, at most 17 minutes, at most 18 minutes, at most 19 minutes, at most 20 minutes, at most 21 minutes, at most 22 minutes, at most 23 minutes, at most 24 minutes, at most 25 minutes, at most 26 minutes, at most 27 minutes, at most 28 minutes, at most 29 minutes, at most 30 minutes, at most 31 minutes, at most 32 minutes, at most 33 minutes, at most 34 minutes, at most 35 minutes, at most 36 minutes, at most 37 minutes, at most 38 minutes, at most 39 minutes, at most 40 minutes, at most 41 minutes, at most 42 minutes, at most 43 minutes, at most 44 minutes, at most 45 minutes, at most 46 minutes, at most 47 minutes, at most 48 minutes, at most 49 minutes, at most 50 minutes, at most 51 minutes, at most 52 minutes, at most 53 minutes, at most 54 minutes, at most 55 minutes, at most 56 minutes, at most 57 minutes, at most 58 minutes, at most 59 minutes, at most 60 minutes, at most 70 minutes, at most 75 minutes, at most 80 minutes, or at most 90 minutes.

Exonuclease treatment

[0094] Embodiments of the various aspects described herein comprise a step of cleaving/digesting one strand of a double-stranded amplicon to produce the single-stranded amplicon. In some embodiments, one strand of the double-stranded amplicon comprises a nucleic acid modification capable of inhibiting 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease, and the step of cleaving/digesting one strand of the double-stranded amplicon comprises contacting the double-stranded amplicon with an exonuclease, e.g., a 5’ to 3’ exonuclease to produce single-stranded amplicons. The strand comprising the nucleic acid modification capable of inhibiting 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease is protected against cleavage. In some embodiments, the other strand of the double-stranded amplicon comprises a 5’ -OH or a nucleic acid modification capable of enhancing the 5’ to 3’ cleaving activity of an exonuclease, for example, a phosphate group at the 5’ -end. [0095] Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3' or the 5' end occurs.

[0096] Exemplary exonucleases amenable to the methods, compositions, kits and systems described herein include, but are not limited to, T7 exonuclease, lambda exonuclease, Exonuclease VIII, T5 exonuclease, and RecJf. In some embodiments of any of the aspects, the exonuclease is T7 exonuclease. T7 exonuclease is a double-stranded DNA specific exonuclease. T7 exonuclease can also be referred to as Exonuclease gp6, Gene product 6 (EC:3.1.11.3), or Gp6. T7 exonuclease initiates at the 5' termini of linear or nicked double- stranded DNA. T7 exonuclease catalyzes the removal of nucleotides from linear or nicked double-stranded DNA in the 5' to 3' direction. T7 Exonuclease can be used for site-directed mutagenesis or nick-site extension.

[0097] In some embodiments of any of the aspects, the T7 exonuclease is isolated or derived from an E. coli strain that carries the cloned T7 exonuclease gene (gene 6) from Escherichia phage T7 (Bacteriophage T7). An exemplary buffer for T7 exonuclease is NEBuffer 4 comprising, for example, Potassium Acetate, Tris-acetate, Magnesium Acetate, and/or DTT.

[0098] In some embodiments of any of the aspects, the exonuclease is lambda exonuclease. Lambda exonuclease can also be referred to as Exodeoxyribonuclease (lambda-induced), EC 3.1.11.3, phage lambda-induced exonuclease, Escherichia coli exonuclease IV, E. coli exonuclease IV, exodeoxyribonuclease IV, and exonuclease IV. Lambda exonuclease has preference for double-stranded DNA (dsDNA), meaning that it degrades a single strand of dsDNA, primarily any strand which has a phosphate at its 5' end. Lambda exonuclease catalyzes the removal of nucleotides from linear or nicked double-stranded DNA in the 5' to 3' direction. Lambda exonuclease exhibits highly processive degradation of double-stranded DNA from the 5' end. The preferred substrate of Lambda exonuclease is 5'-phosphorylated double-stranded DNA, although non-phosphorylated substrates are degraded at a greatly reduced rate. In some embodiments of any of the aspects, Lambda Exonuclease can be used for conversion of double-stranded amplicons to single-stranded amplicons via preferred activity on 5'-phosphorylated ends.

An exemplary buffer for the Lambda exonuclease is Lambda Exonuclease Reaction Buffer comprising, for example, Glycine-KOH, MgCl ₂ , and Bovine Serum Albumin (BSA).

[0099] It is noted that exonuclease can be used in any desired amount for converting double-stranded amplicons to single-stranded amplicons. For example, the exonuclease is provided (i.e., added to the reaction mixture) at a concentration of 0.1 U/ μL to 5 U/ μL. As used herein one unit (e.g., of T7 exonuclease) is defined as the amount of enzyme required to produce 1 nmol of acid-soluble deoxyribonucleotide in a total reaction volume of 50 pi in 30 minutes at 37°C in IX NEBuffer 4 with 0.15 mM sonicated duplex [ ³ H]-DNA; or one unit of an exonuclease, e.g., Lambda exonuclease is defined as the amount of enzyme required to produce 10 nmol of acid-soluble deoxyribonucleotide from double-stranded substrate in a total reaction volume of 50 pi in 30 minutes at 37°C in IX Lambda Exonuclease Reaction Buffer with 1 pg sonicated duplex [ ³ H]-DNA.

[00100] In some embodiments, one strand of the double-stranded amplicon comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more uracil bases, and the step of cleaving/digesting one strand of the double-stranded amplicon comprises contacting the double-stranded amplicon with a uracil-specific endonuclease to produce single-stranded amplicons. For example, the step of cleaving/digesting one strand of the double-stranded amplicon comprises contacting the double-stranded amplicon with USER™ (Uracil-Specific Excision Reagent) enzyme. USER Enzyme generates a single nucleotide gap at the location of a uracil base. USER™ Enzyme is a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII. UDG catalyzes the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. The lyase activity of Endonuclease VIII breaks the phosphodiester backbone at the 3 ^' and 5 ^' sides of the abasic site so that base-free deoxyribose is released. It is noted that the uracil base(s) can be present at the 5’ -terminal of the cleaved/digested strand. Further, if two or more uracil bases are present they can be next to each other or in close proximity to each other. For example, any two uracil bases can be next to each other or within 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions of each other.

[00101] It is noted that double-stranded amplicon where one strand comprises one or more uracil bases can be produced by using a primer comprising one or more uracil bases in the amplification step. Thus, in some embodiments of any one of the aspects described herein, a primer used in the amplification of a target nucleic acid can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more uracil bases. Further, when two or more uracil bases are present they can be next to each other or in close proximity to each other. For example, any two uracil bases can be next to each other or within 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions of each other. [00102] In some embodiments, the uracil bases are present in a region complementary to an index domain of the complementary strand in the double-stranded amplicon.

[00103] It is noted that USER™ Enzyme can be used in any desired amount for converting double-stranded amplicons to single-stranded amplicons. For example, the USER™ Enzyme is provided (i.e., added to the reaction mixture) at a concentration of 0.1 U/μL to 5 U/μL. As used herein one unit of USER™ Enzyme is defined as the amount of enzyme required to nick 10 pmol of a 34-mer double-stranded oligonucleotide comprising a single uracil base, in 15 minutes at 37°C in a IX rCutSmart™ Buffer (50 mM Potassium acetate, 20 mM Tris-acetate, 10 mM Magnesium acetate, 100 pg/ml Recombinant Albumin, pH 7.9) in a total reaction volume of 10 pi.

[00104] In some embodiments of any of the aspects, the double-stranded amplicons are contacted with an exonuclease or USER™ Enzyme at a concentration of from about 0.1 U/μL to about 50 U/μL. For example, the double-stranded amplicons are contacted with an exonuclease or USER™ Enzyme at a concentration of about 0.1 U/μL, about 0.2 U/μL, about 0.3 U/μL, about 0.4 U/μL, about 0.5 U/μL, about 0.6 U/μL, about 0.7 U/μL, about 0.8 U/μL, about 0.9 U/μL, about 1.0 U/μL, about 1.1 U/μL, about 1.2 U/μL, about 1.3 U/μL, about 1.4 U/μL, about 1.5 U/μL, about 1.6 U/μL, about 1.7 U/μL, about 1.8 U/μL, about 1.9 U/μL, about 2.0 U/μL, about 2.1 U/μL, about 2.2 U/μL, about 2.3 U/μL, about 2.4 U/μL, about 2.5 U/μL, about 2.6 U/μL, about 2.7 U/μL, about 2.8 U/μL, about 2.9 U/μL, about 3.0 U/μL, about 3.1 U/μL, about 3.2 U/μL, about 3.3 U/μL, about 3.4 U/μL, about 3.5 U/μL, about 3.6 U/μL, about

3.7 U/μL, about 3.8 U/μL, about 3.9 U/μL, about 4.0 U/μL, about 4.1 U/μL, about 4.2 U/μL, about 4.3 U/μL, about 4.4 U/μL, about 4.5 U/μL, about 4.6 U/μL, about 4.7 U/μL, about 4.8 U/μL, about 4.9 U/μL, about 5.0 U/μL, about 5.1 U/μL, about 5.2 U/μL, about 5.3 U/μL, about 5.4 U/μL, about 5.5 U/μL, about 5.6 U/μL, about 5.7 U/μL, about 5.8 U/μL, about 5.9 U/μL, about 6.0 U/μL, about 6.1 U/μL, about 6.2 U/μL, about 6.3 U/μL, about 6.4 U/μL, about 6.5 U/μL, about 6.6 U/μL, about 6.7 U/μL, about 6.8 U/μL, about 6.9 U/μL, about 7.0 U/μL, about 7.1 U/μL, about 7.2 U/μL, about 7.3 U/μL, about 7.4 U/μL, about 7.5 U/μL, about 7.6 U/μL, about 7.7 U/μL, about 7.8 U/μL, about 7.9 U/μL, about 8.0 U/μL, about 8.1 U/μL, about 8.2 U/μL, about 8.3 U/μL, about 8.4 U/μL, about 8.5 U/μL, about 8.6 U/μL, about 8.7 U/μL, about

8.8 U/μL, about 8.9 U/μL, about 9.0 U/μL, about 9.1 U/μL, about 9.2 U/μL, about 9.3 U/μL, about 9.4 U/μL, about 9.5 U/μL, about 9.6 U/μL, about 9.7 U/μL, about 9.8 U/μL, about 9.9 U/μL, about 10 U/μL, about 20 U/μL, about 30 U/μL, about 40 U/μL, or about 50 U/μL. [00105] The step of contacting the double-stranded amplicons with the exonuclease or USER™ Enzyme can be performed at a temperature between from about 12°C to about 45°C. For example, the step of contacting the double-stranded amplicons with the exonuclease is performed at about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, or about 45°C.

[00106] In some embodiments of any of the aspects, the step of contacting the double- stranded amplicons with the exonuclease or USER™ Enzyme is performed at about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, or about 45°C.

[00107] As a non-limiting example, the step of cleaving one strand of a double-stranded amplicon is performed at a temperature of at least 12°C, at least 13°C, at least 14°C, at least 15°C, at least 16°C, at least 17°C, at least 18°C, at least 19°C, at least 20°C, at least 21°C, at least 22°C, at least 23°C, at least 24°C, at least 25°C, at least 26°C, at least 27°C, at least 28°C, at least 29°C, at least 30°C, at least 31°C, at least 32°C, at least 33°C, at least 34°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 39°C, at least 40°C, at least 41°C, at least 42°C, at least 43°C, at least 44°C, or at least 45°C.

[00108] In some embodiments of any of the aspects, the step of contacting the double- stranded amplicons with the exonuclease or USER™ Enzyme is performed at a temperature of at most 12°C, at most 13°C, at most 14°C, at most 15°C, at most 16°C, at most 17°C, at most 18°C, at most 19°C, at most 20°C, at most 21°C, at most 22°C, at most 23 °C, at most 24°C, at most 25°C, at most 26°C, at most 27°C, at most 28°C, at most 29°C, at most 30°C, at most 31°C, at most 32°C, at most 33°C, at most 34°C, at most 35°C, at most 36°C, at most 37°C, at most 38°C, at most 39°C, at most 40°C, at most 41°C, at most 42°C, at most 43 °C, at most 44°C, or at most 45°C.

[00109] In some embodiments of any of the aspects, the step of contacting the double- stranded amplicons with the exonuclease or USER™ Enzyme is performed at room temperature or ambient temperature (e.g., 20°C-26°C).

[00110] The treatment with the exonuclease or USER™ Enzyme can be for any desired time. For example, the double-stranded amplicons can be contacted with the exonuclease or USER™ Enzyme for a period of from about 15 seconds to about 2 hours. In some embodiments, the treatment with the exonuclease or USER™ Enzyme is for about 1 minutes. As a non-limiting example, the treatment with the exonuclease or U SER™ Enzyme is for about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 60 minutes, about 70 minutes, about 75 minutes, about 80 minutes, or about 90 minutes.

[00111] In some embodiments of any of the aspects, the treatment with the exonuclease or USER™ Enzyme is for at most 5 minutes, at most 6 minutes, at most 7 minutes, at most 8 minutes, at most 9 minutes, at most 10 minutes, at most 11 minutes, at most 12 minutes, at most 13 minutes, at most 14 minutes, at most 15 minutes, at most 16 minutes, at most 17 minutes, at most 18 minutes, at most 19 minutes, at most 20 minutes, at most 21 minutes, at most 22 minutes, at most 23 minutes, at most 24 minutes, at most 25 minutes, at most 26 minutes, at most 27 minutes, at most 28 minutes, at most 29 minutes, at most 30 minutes, at most 31 minutes, at most 32 minutes, at most 33 minutes, at most 34 minutes, at most 35 minutes, at most 36 minutes, at most 37 minutes, at most 38 minutes, at most 39 minutes, at most 40 minutes, at most 41 minutes, at most 42 minutes, at most 43 minutes, at most 44 minutes, at most 45 minutes, at most 46 minutes, at most 47 minutes, at most 48 minutes, at most 49 minutes, at most 50 minutes, at most 51 minutes, at most 52 minutes, at most 53 minutes, at most 54 minutes, at most 55 minutes, at most 56 minutes, at most 57 minutes, at most 58 minutes, at most 59 minutes, at most 60 minutes, at most 70 minutes, at most 75 minutes, at most 80 minutes, or at most 90 minutes.

[00112] In some embodiments, treatment with the exonuclease or USER™ Enzyme is for about 1 minute at room temperature.

[00113] It is noted that amplification and the cleavage/digestion (e.g., exonuclease or USER™ Enzyme treatment) steps can be carried out in the same reaction vessel or in separate reaction vessels. For example, the exonuclease or USER™ Enzyme can be added to the amplification reaction vessel.

[00114] It is noted that the target nucleic acids can be amplified separately and the produced amplicons pooled prior to the cleaving/digestion step. In some other embodiments, the target nucleic acids can be amplified separately and the produced amplicons treated to produce the single-stranded amplicons prior to pooling the single-stranded amplicons. In some embodiments, the target nucleic acids are pooled prior to the amplification.

[00115] In some embodiments of any of the aspects, the method further comprises a step of adding a surfactant to the double-stranded amplicons prior to contacting with the exonuclease or USER™ Enzyme. The surfactant can be in ionic surfactant or a non-ionic surfactant. For example, the surfactant can anionic, cationic or zwitterionic.

[00116] Exemplary anionic surfactants include, but are not limited to, alkyl sulfate, alkyl ether sulfate, alkyl sulfonate, alkylaryl sulfonate, alkyl succinate, alkyl sulfobutane Diacid salt, N-alkylfluorenyl sarcosinate, fluorenyl taurate, fluorenyl isethionate, alkyl phosphate, alkyl ether phosphate, alkyl ether carboxylate, a- Olefin sulfonates and alkali metal salts and alkaline earth metal salts and ammonium salts with their triethanolamine salts. Specific exemplary anionic surfactants include, but are not limited to, ammonium laurylsulfosuccinate, sodium lauryl sulfate, sodium lauryl ether sulfate, ammonium lauryl ether sulfate, triethanolamine dodecylbenzenesulfonate, Sodium lauryl sarcosinate, ammonium lauryl sulfate, sodium oleyl succinate, sodium lauryl sulfate and sodium dodecylbenzenesulfonate. Exemplary cationic surfactants include, but are not limited to, cetylpyridinium chloride, cetyltrimethylammonium bromide (CTAB; Calbiochem #B22633 or Aldrich #85582-0), cetyltrimethylammonium chloride (CTAC1; Aldrich #29273-7), dodecyltrimethylammonium bromide (DTAB, Sigma #D-8638), dodecyltrimethylammonium chloride (DTAC1), octyl trimethyl ammonium bromide, tetradecyltrimethylammonium bromide (TTAB), tetradecyltrimethylammonium chloride (TTAC1), dodecylethyidimethylammonium bromide (DEDTAB), decyltrimethylammonium bromide (DIOTAB), dodecyltriphenylphosphonium bromide (DTPB), octadecylyl trimethyl ammonium bromide, stearoalkonium chloride, olealkonium chloride, cetrimonium chloride, alkyl trimethyl ammonium methosulfate, palmitamidopropyl trimethyl chloride, quaternium 84 (Mackernium NLE; McIntyre Group, Ltd.), and wheat lipid epoxide (Mackernium WLE; McIntyre Group, Ltd.), octyldimethylamine, decyidimethylamine, dodecyidimethylamine, tetradecyldimethylamine, hexadecyidimethylamine, octyldecyldimethylamine, octyidecylmethylamine, didecylmethylamine, dodecylmethylamine, triacetylammonium chloride, cetrimonium chloride, and alkyl dimethyl benzyl ammonium chloride. Additional classes of cationic surfactants include, but are not limited to, phosphonium, imidzoline, and ethylated amine groups.

[00117] In some embodiments of the various aspects, the surfactant is an anionic surfactant. [00118] In some preferred embodiments of any of the aspects, the surfactant is SDS. [00119] The surfactant can be added to any desired amount. For example, the surfactant can be added to a final concentration of about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15, mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM.

[00120] In some embodiments of any of the aspects, the method further comprises a step of heating the double-stranded amplicons prior to contacting with the exonuclease or USER™ Enzyme. Without wishing to be bound by a theory, the heating step can inactivate the enzymes (e.g., polymerase, recombinase, etc.) used for the amplification. Generally, the double- stranded amplicons can be heated to a temperature from about 40°C to about 95°C prior to exonuclease treatment. For example, the double-stranded amplicons can be heated to a temperature of about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about

53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about

68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, about 75°C, about 76°C, about 77°C, about 78°C, about 79°C, about 80°C, about 81°C, about 82°C, about

83°C, about 84°C, about 85°C, about 86°C, about 87°C, about 88°C, about 89°C, about 90°C, about 91°C, about 92°C, about 93°C, about 94°C, or about 95°C.

[00121] In some embodiments of any of the aspects, the double-stranded amplicons are heated for a period of from about 15 seconds to about 2 hours. For example, the double- stranded amplicons can be heated for a period of about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 60 minutes, about 70 minutes, about 75 minutes, about 80 minutes, or about 90 minutes.

Detection of single-stranded amplicons

[00122] The single-stranded amplicons can be detected by means that can distinguish a first single-stranded amplicon from a different second single-stranded amplicon. Detection methods can include, but are not limited to, enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.

[00123] Any of a number of different assays can be used to detect the single-stranded amplicons as described herein. In some embodiments of any of the aspects, the detection method is selected from the group consisting of: lateral flow detection; hybridization with conjugated or unconjugated DN A; colorimetric assays; gel electrophoresis; a toehold-mediated strand displacement reaction; molecular beacons; fluorophore-quencher pairs; microarrays; Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK); DNA endonuclease-targeted CRISPR trans reporter (DETECTR); sequencing; and quantitative polymerase chain reaction (qPCR). In some embodiments of any of the aspects, the detection method comprises a plate-based assay (e.g., SHERLOCK, DETECTR, microarray, hybridization, qPCR, sequencing, etc.). In some embodiments of any of the aspects, the detection method comprises a lateral flow assay. In some embodiments of any of the aspects, the detection method is colorimetric, luminescent, or fluorescent, etc.

[00124] In some embodiments of any of the aspects, the single-stranded amplicons are detected using lateral flow detection, also known as a lateral flow immunoassay test (LFIA), laminar flow, the immunochromatographic assay, or strip test. LFIAs are a simple device intended to detect the presence (or absence) of antigen, e.g. a nucleic acid or polypeptide, in a fluid sample. There are currently many LFIA tests used for medical diagnostics, either for home testing, point of care testing, or laboratory use. LFIA tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test strip it encounters a colored reagent (generally comprising antibody specific for the test target antigen) bound to microparticles which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with an antibody (e.g., specific for a detectable marker on the target nucleic acid or for a detectable marker on a complementary nucleic to the target nucleic acid) or pretreated with a conjugated or unconjugated DNA as described herein. Depending upon the level of target present in the sample the colored reagent can be captured and become bound at the test line or zone. LFIAs are essentially immunoassays adapted to operate along a single axis to suit the test strip format or a dipstick format. Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens from fluid samples such as urine, blood, water, and/or homogenized tissue samples etc. Strip tests are also known as dip stick tests, the name bearing from the literal action of "dipping" the test strip into a fluid sample to be tested. LFIA strip tests are easy to use, require minimum training and can easily be included as components of point-of-care test (POCT) diagnostics to be use on site in the field. LFIA tests can be operated as either competitive or sandwich assays. Sandwich LFIAs are similar to sandwich ELISA. The sample first encounters colored particles which are labeled with antibodies raised to the target antigen. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the antigen. The test line will show as a colored band in positive samples. In some embodiments of any of the aspects, the lateral flow immunoassay can be a double antibody sandwich assay, a competitive assay, a quantitative assay or variations thereof. Competitive LFIAs are similar to competitive ELISA. The sample first encounters colored particles which are labeled with the target antigen or an analogue. The test line contains antibodies to the target/its analogue. Unlabeled antigen in the sample will block the binding sites on the antibodies preventing uptake of the colored particles. The test line will show as a colored band in negative samples. There are a number of variations on lateral flow technology. It is also possible to apply multiple capture zones to create a multiplex test.

[00125] The use of lateral flow tests to detect nucleic acids have been described in the art; see e.g., U.S. Pat. Nos. 9,121,849; 9,207,236; and 9,651,549; the content of each of which is incorporated herein by reference in its entirety. The use of "dip sticks" or LFIA test strips and other solid supports have been described in the art in the context of an immunoassay for a number of targets. U.S. Pat. Nos. 4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871; 6,565,808, U. S. patent applications Ser. No. 10/278,676; U.S. Ser. No. 09/579,673 and U.S. Ser. No. 10/717,082, which are incorporated herein by reference in their entirety, are nonlimiting examples of such lateral flow test devices. Examples of patents that describe the use of "dip stick" technology to detect soluble antigens via immunochemical assays include, but are not limited to US PatentNos. 4,444,880; 4,305,924; and 4,135,884; which are incorporated by reference herein in their entireties. The apparatuses and methods of these three patents broadly describe a first component fixed to a solid surface on a "dip stick" which is exposed to a solution containing a soluble antigen that binds to the component fixed upon the "dip stick," prior to detection of the component-antigen complex upon the stick.

[00126] While use of lateral flow assays is known in the art, they do not allow multiplex detection of different antigens. The detection methods described herein overcome this deficiency of lateral flow assays known in the art. Accordingly, in some embodiments of any of the aspects, the detection step comprises hybridizing the single-stranded amplicons with a first nucleic acid probe and a second nucleic acid probe to form a plurality of complexes. The first nucleic acid probe comprises a detection ligand and a nucleotide sequence substantially complementary to a first region of a member of the plurality of single-stranded amplicons. The second nucleic acid probe comprises a capture ligand and a nucleotide sequence substantially complementary to a second region of the member of the plurality. The detection ligand and the capture ligand are different. Further, the capture ligand of the second nucleic acid probe in a first complex of the plurality of complexes is distinguishable from the capture ligand of the second nucleic acid probe in a second complex of the plurality of the complexes. [00127] The complexes comprising the single-stranded amplicon and the first and second nucleic acid probes are applied to a lateral flow the test strip. The test strip comprises plurality of test regions. Each test regions comprises a ligand binding molecule immobilized therein. The ligand binding molecule in each test region is capable of binding the capture ligand of the second nucleic acid probe in the complex and the ligand binding molecules in different test regions bind different capture ligands. For example, the ligand binding molecule in a first test region is capable of binding with the capture ligand in a first complex and the ligand binding molecule in a second test region is capable of binding with the capture ligand in a second complex.

[00128] Binding of the capture ligand with the ligand binding molecule in the test region immobilizes the complex in the test regions. As ligand binding molecules bind with different capture ligands, different the complexes are immobilized in different test regions. The immobilized complexes then can be detected using a ligand binding molecule comprising a detectable label and capable of binding the detection ligand on the first nucleic acid probe. [00129] In some embodiments of any of the aspects, the detection step comprises hybridizing the single-stranded amplicons with a nucleic acid probe to form a plurality of complexes. Each nucleic acid probe independently comprises a detection ligand. Nucleic acid probes independently comprise a nucleotide sequence substantially complementary to one of the single-stranded amplicons. For example, a first nucleic acid probe comprises a nucleotide sequence substantially complementary to a first region of a first member of the plurality of single-stranded amplicons and a second nucleic acid probe comprises a nucleotide sequence substantially complementary to a first region of a second member of the plurality of single- stranded amplicons.

[00130] The complexes comprising the single-stranded amplicon and the nucleic acid probe are applied to a lateral flow test strip. The test strip comprises plurality of test regions. Each test region comprises a nucleic acid capture probe therein. The nucleic acid capture probe in each test region is capable of hybridizing with the single-stranded amplicon in the complex, and the nucleic acid capture probes in different test regions hybridize with different single- stranded amplicons. For example, the nucleic acid capture probe in a first test region is capable of hybridizing with a single-stranded amplicon in a first complex and the nucleic acid capture probe in second test region is capable of hybridizing with a single-stranded amplicon in a second complex.

[00131] Hybridization of the single-stranded amplicon capture probe with a nucleic acid probe in a test region immobilizes the complex in the test regions. As capture probes hybridize with different single-stranded amplicons, different the complexes are immobilized in different test regions. The immobilized complexes can be detected using a ligand binding molecule comprising a detectable label and capable of binding the ligand on the nucleic acid probe hybridized with the single-stranded amplicon.

[00132] In some embodiments, the single-stranded amplicons comprise a detection ligand and the single-stranded amplicons are applied to a lateral flow the test strip. The test strip comprises plurality of test regions. Each test regions comprises a nucleic acid capture probe therein. The nucleic acid capture probe in each test region are capable of hybridizing with the single-stranded amplicon in the complex and the nucleic acid capture probes in different test regions hybridize with different single-stranded amplicons. For example, the nucleic acid capture probe in a first test region is capable of hybridizing with a first single- stranded amplicon and the nucleic acid capture probe in second first test region is capable of hybridizing with a second (e.g., different) single-stranded amplicon.

[00133] Hybridization of the single-stranded amplicon with a nucleic acid probe in a test region immobilizes the single-stranded amplicon in the test regions. As capture probes hybridize with different single-stranded amplicons, different single-stranded amplicons are immobilized in different test regions. The immobilized single-stranded amplicons can be detected using the detection ligand attached to the single-stranded amplicon. For example, the detection ligand can be a detectable label. Alternatively, the immobilized single-stranded amplicons can be detected using a ligand binding molecule comprising a detectable label and capable of binding the detection ligand on the amplicon.

[00134] It is noted that a nucleic acid probe, e.g. the first nucleic acid probe, the second nucleic acid probe and/or the capture probe can hybridize at an inner region of the single- stranded amplicon. As used herein, the term “inner region” refers to a region of the amplicon that does not comprise a primer binding-site. In some embodiments of any of the aspects, the first nucleic acid probe hybridizes at an inner region of the single-stranded amplicon. In some embodiments of any of the aspects, the second nucleic acid probe hybridizes at an inner region of the single-stranded amplicon. In some embodiments of any of the aspects, the capture probe hybridizes at an inner region of the single-stranded amplicon. In some embodiments of any of the aspects, the first and second nucleic acid probes hybridize at an inner region of the single- stranded amplicon. In some embodiments of any of the aspects, the first nucleic acid probe and the capture probe hybridizes at an inner region of the single-stranded amplicon.

[00135] In some embodiments, the single-stranded amplicon comprises an index domain and the second nucleic acid probe and/or the capture probe can hybridize with the index domain of the. The capture/test regions in the lateral flow the test strip can be indexed and used to identified the single-stranded amplicons from different target nucleic acids or samples.

[00136] The ligands, e.g., the detection ligand and the capture ligand can be independently selected from the group consisting of organic and inorganic molecules, peptides, polypeptides, proteins, peptidomimetics, glycoproteins, lectins, nucleosides, nucleotides, monosaccharides, di saccharides, trisaccharides, oligosaccharides, polysaccharides, lipopolysaccharides, vitamins, steroids, hormones, cofactors, receptors, receptor ligands, and analogs and derivatives thereof.

[00137] In some embodiments of any of the aspects, the capture ligands and/or the detection ligands are independently one member of a binding pair.

[00138] As used herein, the term “binding pair” refers to a pair of moieties that specifically bind each other with high affinity, generally in the low micromolar to picomolar range. When one member of a binding pair is conjugated to a first element and the other member of the pair is conjugated to a second element, the first and second elements will be brought together by the interaction of the members of the binding pair. Non-limiting examples of binding pairs include antigemantibody (including antigen-binding fragments or derivatives thereof), biotin: avi din, biotin: streptavi din, biotinmeutravidin (or other variants of avi din that bind biotin), receptor: ligand, and the like. Additional molecule for binding pair can include, neutravidin, strep-tag, strep-tactin and derivatives, and other peptide, hapten, dye-based tags-anti-Tag combinations such as SpyCatcher-SpyTag, His-Tag, Fc Tag, Digitonin, GFP, FAM, SNAP- TAG. HRP, FLAG, HA, myc, glutathione S-transferase (GST), maltose binding protein (MBP), small molecules, and the like.

[00139] In some embodiments of any of the aspects, the capture ligands are independently an antigen.

[00140] In some embodiments of any of the aspects, the detection ligands are independently a detectable label. For example, the detection ligands are independently a fluorophore, e.g., fluorescein.

[00141] Embodiments of the various aspects described herein include ligand binding molecules. As used herein, the term “ligand binding molecule” refers to a molecule that binds specifically to given ligand. As used herein, the terms “binds specifically”, and “binding specificity” in reference to a ligand binding molecule refers to its capacity to bind to a given target ligand preferentially over other non-target ligands. For example, if the ligand binding molecule (“molecule A”) is capable of “binding specifically” to a given target ligand (“molecule B”), molecule A has the capacity to discriminate between molecule B and any other number of potential alternative binding partners. Accordingly, when exposed to a plurality of different but equally accessible molecules as potential binding partners, molecule A will selectively bind to molecule B and other alternative potential binding partners will remain substantially unbound by molecule A. In general, molecule A will preferentially bind to molecule B at least 10-fold, preferably 50-fold, more preferably 100-fold, and most preferably greater than 100-fold more frequently than other potential binding partners. Molecule A may be capable of binding to molecules that are not molecule B at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from molecule B-specific binding, for example, by use of an appropriate control.

[00142] By way of non-limiting example, the ligand binding molecules can be one member of a binding pair. For example, the ligand binding molecules can be independently selected antibodies. In some embodiments of any of the aspects, the ligand binding molecules are independently selected from the group consisting of: anti-FAM antibodies, anti-digoxigenin antibodies, anti-tetramethylrhodamine (TAMRA) antibodies, anti-Texas Red antibodies, anti- dinitrophenyl antibodies, anti-cascade blue antibodies, anti-streptavidin antibodies, anti-biotin antibodies, anti-Cy5 antibodies, anti-dansyl antibodies, anti-fluorescein antibodies, streptavidin and biotin.

[00143] In some embodiments of any of the aspects, the ligand molecule capable of binding with a capture ligand is an antibody. [00144] In some embodiments of any of the aspects, the ligand molecule capable of binding with a detection ligand is an antibody comprising a detectable label.

[00145] In some embodiments of any of the aspects, one or more of the detection reagents (e.g. an antibody reagent and/or nucleic acid probe) can comprise a detectable label and/or comprise the ability to generate a detectable signal (e.g. by catalyzing a reaction converting a compound to a detectable product). As used herein, the term “detectable label” or “detectable marker” refers to a molecule or composition capable of producing a detectable signal indicative of the presence of a target. Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g. antibodies and nucleic acid probes) are well known in the art.

[00146] In some embodiments of any of the aspects, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluorescence, or chemiluminescence, or any other appropriate means. The detectable labels described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies). The detectable label can be linked by covalent or non-covalent means to the reagent. Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.

[00147] In other embodiments, a detection reagent (e.g., a primer, a probe, etc.) is labeled with a fluorescent compound. When the fluorescently labeled reagent is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. In some embodiments of any of the aspects, a detectable label can be a fluorescent dye molecule, or fluorophore. A wide variety of fluorescent reporter dyes are known in the art. Typically, the fluorophore is an aromatic or heteroaromatic compound and can be a pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothi azole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine or other like compound. Exemplary fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS ; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein (pH 10); 5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5- TAMRA (5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7- Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2- methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC- Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); BG- 647; Bimane; Bisbenzamide; Blancophor FFG; Blancophor SV; BOBO™ -1; BOBO™ -3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy FI; Bodipy FL ATP; Bodipy Fl- Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -1; BO-PRO™ -3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green- 1 Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CFDA; CFP - Cyan Fluorescent Protein; Chlorophyll; Chromomycin A; Chromomycin A; CMFDA; Coelenterazine ; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine O; Coumarin Phalloidin; CPM Methylcoumarin; CTC; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); d2; Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Diehl orodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4- ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP); DIDS; Dihydorhodamine 123 (DHR); DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium homodimer- 1 (EthD-1); Euchrysin; Europium (III) chloride; Europium; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FITC; FL-645; Flazo Orange; Fluo-3; Fluo-4; Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1- 43™; FM 4-46; Fura Red™ (high pH); Fura-2, high calcium; Fura-2, low calcium; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; Lucifer Yellow; Mag Green; Magdala Red (Phloxin B); Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant Iavin E8G; Oregon Green™; Oregon Green 488-X; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 ; PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufm; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200 ; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R- phycoerythrin (PE); red shifted GFP (rsGFP, S65T); S65A; S65C; S65L; S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™; sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (super glow GFP); SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SPQ (6-methoxy- N-(3-sulfopropyl)-quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine G Extra; Tetracycline; Tetramethylrhodamine ; Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO- PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5); TRITC (TetramethylRodaminelsoThioCyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3. Many suitable forms of these fluorescent compounds are available and can be used. Additional fluorophore examples include, but are not limited to fluorescein, phycoerythrin, phycocyanin, o- phthalaldehyde, fluorescamine, Cy3™, Cy5™, allophycocyanin, Texas Red, peridinin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas red and tetramethylrhodamine isothiocyanate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™, 6-carboxyfluorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 6-carboxy- 4',5'-dichloro-2',7'-dimethoxyfiuorescein (JOE or J), N,N,N',N'-tetramethyl- 6carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5- carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g., umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g., cyanine dyes such as Cy3, Cy5, etc.; BODIPY dyes and quinoline dyes.

[00148] Other exemplary detectable labels include luminescent and bioluminescent markers (e.g., biotin, luciferase (e.g, bacterial, firefly, click beetle and the like), luciferin, and aequorin), radiolabels (e.g, 3H, 1251, 35S, 14C, or 32P), enzymes (e.g, galactosidases, glucorinidases, phosphatases (e.g, alkaline phosphatase), peroxidases (e.g, horseradish peroxidase), and cholinesterases), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g, polystyrene, polypropylene, and latex) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which is incorporated herein by reference.

[00149] Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photo-detector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with an enzyme substrate and detecting the reaction product produced by the action of the enzyme on the enzyme substrate, and calorimetric labels can be detected by visualizing the colored label.

[00150] In some embodiments of any of the aspects, a detectable label can be a radiolabel including, but not limited to ³ H, ¹²⁵ 1, ³⁵ S, ¹⁴ C, ³² P, and ³³ P. Suitable non-metallic isotopes include, but are not limited to, ¹¹ C, ¹⁴ C, ¹³ N, ¹⁸ F, ¹²³ I, ¹²⁴ I, and ¹²⁵ I. Suitable radioisotopes include, but are not limited to, "mTc, ⁹⁵ Tc, ^{1 1} ln, ⁶² Cu, ⁶⁴ Cu, Ga, ⁶⁸ Ga, and ¹⁵³ Gd. Suitable paramagnetic metal ions include, but are not limited to, Gd(III), Dy(III), Fe(III), and Mn(II). Suitable X-ray absorbers include, but are not limited to, Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Au, Yb, Dy, Cu, Rh, Ag, and Ir.

[00151] In some embodiments, the radionuclide is bound to a chelating agent or chelating agent-linker attached to probe, primer or reagent. Exemplary chelating agents include, but are not limited to, diethylenetriaminepentaacetic acid (DTP A) and ethylenediaminetetraacetic acid (EDTA). Suitable radionuclides for direct conjugation include, without limitation, ³ H, ¹⁸ F, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ³⁵ S, ¹⁴ C, ³² P, and ³³ P and mixtures thereof. Suitable radionuclides for use with a chelating agent include, without limitation, ⁴⁷ Sc, ⁶⁴ Cu, ⁶⁷ Cu, ⁸⁹ Sr, ⁸⁶ Y, ⁸⁷ Y, ⁹⁰ Y, ¹⁰⁵ Rh, ^lu Ag, ¹¹¹ In, ¹¹⁷ mSn, ¹⁴⁹ Pm, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹¹ At, ²¹² Bi, and mixtures thereof. Suitable chelating agents include, but are not limited to, DOTA, BAD, TETA, DTP A, EDTA, NTA, HDTA, their phosphonate analogs, and mixtures thereof. One of skill in the art will be familiar with methods for attaching radionuclides, chelating agents, and chelating agent-linkers to molecules such nucleic acids.

[00152] In some embodiments of any of the aspects, a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose- Vl-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In some embodiments of any of the aspects, a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. In some embodiments of any of the aspects, a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads. [00153] In some embodiments of any of the aspects, detection reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin- peroxidase conjugate and a chromogenic substrate. Such streptavidin peroxidase detection kits are commercially available, e.g., from DAKO; Carpinteria, CA. A reagent can also be detectably labeled using fluorescence emitting metals such as ¹⁵² Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA). [00154] In some embodiments of any of the aspects, the detectable label is selected from the group consisting of a light-absorbing dye, a fluorescent dye, a luminescent or bioluminescent molecule, a quantum dot, a radiolabel, an enzyme, a colorimetric label. In some embodiments of any of the aspects, the detectable label is a colorimetric label selected from the group consisting of colloidal gold, colored glass or plastic beads, and any combinations thereof. In some embodiments of any of the aspects, the detectable label is a gold nanoparticle or a latex bead.

Lateral flow test strips

[00155] Generally, the lateral flow strip comprises a lateral flow matrix comprising a plurality of test regions (also referred to as capture zones herein, a sample receiving zone). It is noted that the terms “lateral flow test strips” and “lateral flow device” are used interchangeably herein. In some embodiments, each capture zone independently comprises a ligand binding molecule immobilized on the lateral flow matrix, wherein the ligand binding molecule is capable of binding a capture ligand, and wherein the ligand binding molecule in one capture zone binds a ligand that is different from the ligand for the ligand binding molecule in a different capture zone. In other words, each capture zone independent comprises a capture ligand immobilized on the lateral flow matrix, and wherein the ligand binding molecule in at least one capture zone binds a capture ligand that is different from the capture ligand bound by a ligand binding molecule in at least one other capture zone.

[00156] In some other embodiments, each capture zone independently comprises a nucleic acid capture probe immobilized on the lateral flow matrix, wherein the capture probe is capable of hybridizing with single-stranded amplicon, and wherein the capture probe in one capture zone hybridizes with a single-stranded amplicon that is different from the single-stranded amplicon that hybridizes with a capture probe in a different zone. In other words, each capture zone independently comprises a nucleic acid capture probe immobilized on the lateral flow matrix, and wherein the capture probe in at least one capture zone is different from the capture probe in at least one other capture zone.

[00157] In some embodiments of any of the aspects, the test strip also comprises a control region or zone. Generally, the control region comprises a ligand binding molecule capable of binding the ligand binding molecule that binds with a detection ligand.

[00158] Generally, the lateral flow strip comprises a lateral flow matrix which defines a flow path and which comprises in series: a sample receiving zone; a plurality of serially oriented capture zones; and, optionally, a control zone. However, other geometric arrangements can also be used. Some exemplary arrangements are shown in Figure 8.

[00159] In some embodiments of any one of the aspects, at least two of the capture zones in the lateral flow test strip are arranged in a predetermined pattern.

[00160] In some embodiments of any one of the aspects, at least two of the capture zones in the lateral flow test strip are configured to capture/detect the same target molecule. Lateral test strip comprising at least two capture zones configured to capture/detect the same target molecule, e.g., single-stranded amplicon, are useful in multiplex detection. The single- stranded amplicons comprising an index domain are captured on two or more capture zones. This allows a practitioner to combinatorially encode a potential 2 ⁿ distinct samples into n spots on the lateral flow test strip. See , for example, FIGS. 6A-6E.

[00161] In some embodiments of any one of the aspects, at least two of the capture zones in the lateral flow test strip are configured to capture/detect different target molecules and at least two of the capture zones in the lateral flow test strip are configured to capture/detect the same target molecule.

[00162] In some embodiments of any one of the aspects, at least two of the capture zones in the lateral flow test strip are configured to capture/detect different target molecules, at least two of the capture zones in the lateral flow test strip are configured to capture/detect the same target molecule, and at least two of the captures zones on the lateral flow test strip are arranged in a predetermined pattern. For example, the at least two of the capture zones configured to capture/detect the same target molecule are arranged in a predetermined pattern and/or the at least two of the capture zones configured to capture/detect different target molecule are arranged in a predetermined pattern.

[00163] In some embodiments of any one of the aspects, at least one capture zone in the lateral flow test strip is configured to capture/detect two different target molecules. [00164] In embodiments of the test strip, the sample receiving zone comprises: (i) a labeling zone comprising diffusively bound nucleic acid probes, e.g., first nucleic acid probe, second nucleic acid probe, and/or capture probe; and (ii) a sample zone for receiving a liquid sample comprising the single-stranded amplicons. Optionally, the labeling zone is positioned between the plurality of capture zones and the sample zone for receiving the liquid sample comprising the single-stranded amplicons.

[00165] It is noted that each capture zone and/or control zone independently has a regular or irregular shape. For example, optionally at least one of the capture zones has a shape selected from the group consisting of a line, a circle, a rod, and a polygonal. In some embodiments, the polygonal is a square, a triangle or a rectangle. In some embodiments, at least two captures zones are of same shape. In some embodiments, at least two captures zones are the same size.

[00166] In some embodiments of any of the aspects, the detection step comprises contacting the single-stranded amplicons with the sample receiving zone of the test strip. The single- stranded amplicons flow to the labeling zone where they hybridize with the appropriate probes and flow to the capture zones.

Target Nucleic Acids

[00167] The target nucleic acids in the plurality can be any desired nucleic acid. Further, the target nucleic acids can be naturally occurring or synthetic nucleic acids. Thus, in some embodiments, the target nucleic acids are naturally occurring nucleic acids. A naturally occurring nucleic acid include a nucleic acid isolated and/or purified from a natural source. [00168] In some embodiments of the various aspects described herein, the target nucleic acid is DNA, e.g., a target DNA. Exemplary target DNAs include, but are not limited to, genomic DNA, viral DNA, cDNA, single-stranded DNA, double-stranded DNA, circular DNA, etc... In some embodiments of the various aspects described herein, the target nucleic acid is an RNA, e.g., a target RNA. Generally, the RNA can be any known type of RNA. In some embodiments of the various aspects described herein, the target RNA is messenger RNA, ribosomal RNA, Signal recognition particle RNA, Transfer RNA, Transfer-messenger RNA, Small nuclear RNA, Small nucleolar RNA, SmYRNA, Small Cajal body-specific RNA, Guide RNA, Ribonuclease P, Ribonuclease MRP, Y RNA, Telomerase RNA Component, Spliced Leader RNA, Antisense RNA, Cis-natural antisense transcript, CRISPR RNA, Long noncoding RNA, MicroRNA, Pi wi -interacting RNA, Small interfering RNA, Short hairpin RNA, Trans-acting siRNA, Repeat associated siRNA, 7SK RNA, Enhancer RNA, Parasitic RNAs, Type, Retrotransposon, Viral genome, Viroid, Satellite RNA, or Vault RNA.

[00169] In some embodiments of the various aspects described herein, the target RNA can be a viral RNA. As used herein, the term “RNA virus” refers to a virus comprising an RNA genome. In some embodiments of the various aspects described herein, the RNA virus is a double-stranded RNA virus, a positive-sense RNA virus, a negative-sense RNA virus, or a reverse transcribing virus (e.g., retrovirus).

[00170] In some embodiments of the various aspects described herein, the RNA virus is a Group III (i.e., double stranded RNA (dsRNA)) virus. In some embodiments of the various aspects described herein, the Group III RNA virus belongs to a viral family selected from the group consisting of: Amalgaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Endomaviridae, Hypoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae (e.g., Rotavirus), Totiviridae, Quadriviridae. In some embodiments of the various aspects described herein, the Group III RNA virus belongs to the Genus Botybirnavirus. In some embodiments of the various aspects described herein, the Group III RNA virus is an unassigned species selected from the group consisting of: Botrytis porri RNA virus 1, Circulifer tenellus virus 1, C oil etotri chum camelliae filamentous virus 1, Cucurbit yellows associated virus, Sclerotinia sclerotiorum debilitation-associated virus, and Spissistilus festinus virus 1.

[00171] In some embodiments of the various aspects described herein, the RNA virus is a Group IV (i.e., positive-sense single stranded (ssRNA)) virus. In some embodiments of the various aspects described herein, the Group IV RNA virus belongs to a viral order selected from the group consisting of: Nidovirales, Picornavirales, and Tymovirales. In some embodiments of the various aspects described herein, the Group IV RNA virus belongs to a viral family selected from the group consisting of: Arteriviridae, Coronaviridae (e.g., Coronavirus, SARS-CoV), Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Mamaviridae, Picornaviridae (e.g., Poliovirus, Rhinovirus (a common cold virus), Hepatitis A virus), Secoviridae (e.g., sub Comovirinae), Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Alphatetraviridae, Alvernaviridae, Astroviridae, Bamaviridae, Benyviridae, Bromoviridae, Caliciviridae (e.g., Norwalk virus), Carmotetraviridae, Closteroviridae, Flaviviridae (e.g., Yellow fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Zika virus), Fusariviridae, Hepeviridae, Hypoviridae, Leviviridae, Luteoviridae (e.g., Barley yellow dwarf virus), Polycipiviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Potyviridae, Sarthroviridae, Statovirus, Togaviridae (e.g., Rubella virus, Ross River virus, Sindbis virus, Chikungunya virus), Tombusviridae, and Virgaviridae. In some embodiments of the various aspects described herein, the Group IV RNA virus belongs to a viral genus selected from the group consisting of: Bacillariornavirus, Dicipivirus, Labyrnavirus, Sequiviridae, Blunervirus, Cilevirus, Higrevirus, Idaeovirus, Negevirus, Ourmiavirus, Polemovirus, Sinaivirus, and Sobemovirus. In some embodiments of the various aspects described herein, the Group IV RNA virus is an unassigned species selected from the group consisting of: Acyrthosiphon pisum virus, Bastrovirus, Blackford virus, Blueberry necrotic ring blotch virus, Cadicistrovirus, Chara australis virus, Extra small virus, Goji berry chlorosis virus, Hepelivirus, Jingmen tick virus, Le Blanc virus, Nedicistrovirus, Nesidiocoris tenuis virus l, Niflavirus, Nylanderiafulva virus 1, Orsay virus, Osedaxjaponicus RNA virus 1, Picalivirus, Plasmopara halstedii virus, Rosellinia necatrix fusarivirus 1, Santeuil virus, Secalivirus, Solenopsis invicta virus 3, Wuhan large pig roundworm virus. In some embodiments of the various aspects described herein, the Group IV RNA virus is a satellite virus selected from the group consisting of: Family Sarthroviridae, Genus Albetovirus, Genus Aumaivirus, Genus Papanivirus, Genus Virtovirus, and Chronic bee paralysis virus.

[00172] In some embodiments of the various aspects described herein, the RNA virus is a Group V (i.e., negative-sense ssRNA) virus. In some embodiments of the various aspects described herein, the Group V RNA virus belongs to a viral phylum or subphylum selected from the group consisting of: Negarnaviricota, Haploviricotina, and Polyploviricotina. In some embodiments of the various aspects described herein, the Group V RNA virus belongs to a viral class selected from the group consisting of: Chunqiuviricetes, Ellioviricetes, Insthoviricetes, Milneviricetes, Monjiviricetes, and Yunchangviricetes. In some embodiments of the various aspects described herein, the Group V RNA virus belongs to a viral order selected from the group consisting of: Articulavirales, Bunyavirales, Goujianvirales, Jingchuvirales, Mononegavirales, Muvirales, and Serpentovirales. In some embodiments of the various aspects described herein, the Group V RNA virus belongs to a viral family selected from the group consisting of: Amnoonviridae (e.g., Taastrup virus), Arenaviridae (e.g., Lassa virus), Aspiviridae, Bomaviridae (e.g., Boma disease virus), Chuviridae, Cruliviridae, Feraviridae, Filoviridae (e.g., Ebola virus, Marburg virus), Fimoviridae, Hantaviridae, Jonviridae, Mymonaviridae, Nairoviridae, Nyamiviridae, Orthomyxoviridae (e.g., Influenza viruses), Paramyxoviridae (e.g., Measles virus, Mumps virus, Nipah virus, Hendra virus, and NDV), Peribunyaviridae, Phasmaviridae, Phenuiviridae, Pneumoviridae (e.g., RSV and Metapneumovirus), Qinviridae, Rhabdoviridae (e.g., Rabies virus), Sunviridae, Tospoviridae, and Yueviridae. In some embodiments of the various aspects described herein, the Group V RNA virus belongs to a viral genus selected from the group consisting of: Anphevirus, Arlivirus, Chengtivirus, Crustavirus, Tilapineviridae, Wastrivirus, and Deltavirus (e.g., Hepatitis D virus).

[00173] In some embodiments of the various aspects described herein, the RNA virus is a Group VI RNA virus, which comprise a virally encoded reverse transcriptase. In some embodiments of the various aspects described herein, the Group VI RNA virus belongs to the viral order Ortervirales. In some embodiments of the various aspects described herein, the Group VI RNA virus belongs to a viral family or subfamily selected from the group consisting of: Belpaoviridae, Caulimoviridae, Metaviridae, Pseudoviridae, Retroviridae (e.g., Retroviruses, e.g. HIV), Orthoretrovirinae, and Spumaretrovirinae. In some embodiments of the various aspects described herein, the Group VI RNA virus belongs to a viral genus selected from the group consisting of: Alpharetrovirus (e.g., Avian leukosis virus; Rous sarcoma virus), Betaretrovirus (e.g., Mouse mammary tumour virus), Bovispumavirus (e.g., Bovine foamy virus), Deltaretrovirus (e.g., Bovine leukemia virus; Human T-lymphotropic virus), Epsilonretrovirus (e.g., Walleye dermal sarcoma virus), Equispumavirus (e.g., Equine foamy virus), Felispumavirus (e.g., Feline foamy virus), Gammaretrovirus (e.g., Murine leukemia virus; Feline leukemia virus), Lentivirus (e.g., Human immunodeficiency virus 1; Simian immunodeficiency virus; Feline immunodeficiency virus), Prosimiispumavirus (e.g., Brown greater galago prosimian foamy virus), and Simiispumavirus (e.g., Eastern chimpanzee simian foamy virus).

[00174] In some embodiments of the various aspects described herein, the RNA virus is selected from influenza virus, human immunodeficiency virus (HIV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments of the various aspects described herein, the RNA virus is influenza virus. In some embodiments of the various aspects described herein, the RNA virus is immunodeficiency virus (HIV). In some embodiments of the various aspects described herein, the RNA virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[00175] In some embodiments of the various aspects described herein, the viral RNA is an RNA produced by a virus with a DNA genome, i.e., a DNA virus. As a non-limiting example the DNA virus is a Group I (dsDNA) virus, a Group II (ssDNA) virus, or a Group VII (dsDNA- RT) virus

[00176] In some embodiments of any one of the aspects, at least one member of the plurality of target nucleic acids is single-stranded.

[00177] In some embodiments of any one of the aspects, at least one member of the plurality of target nucleic acids is double-stranded. [00178] In some embodiments of any one of the aspects, at least one member of the plurality of target nucleic acids is RNA.

[00179] In some embodiments of any one of the aspects, at least one member of the plurality of target nucleic acids is DNA.

[00180] In some embodiments of any one of the aspects, at least one member of the plurality of target nucleic acids is a viral nucleic acid.

[00181] In some embodiments of any one of the aspects, at least one member of the plurality of target nucleic acids is a first viral nucleic acid and at least one member of the plurality of target nucleic acids is a second viral nucleic acid. For example, the first and second viral nucleic acids are from different viruses.

[00182] In some embodiments of any one of the aspects, at least one member of the plurality of target nucleic acids is a viral RNA.

[00183] In some embodiments of any one of the aspects, at least one member of the plurality of target nucleic acids is a viral DNA.

[00184] In some embodiments of the various aspects described herein, the target nucleic acid can be comprised in a sample or test sample. The term “sample” or “test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a subject in need of testing. Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample: blood; serum; plasma; urine; semen; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva, and/or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term “test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments of the various aspects described herein, a test sample can comprise cells from a subject.

[00185] In some embodiments of the various aspects described herein, the sample can include a viral transport media (VTM). Non-limiting examples of viral transport media include COPAN Universal Transport Medium; Eagle Minimum Essential Medium (E-MEM); Transport medium 199; and PBS-Glycerol transport medium. See e.g., Johnson, Transport of Viral Specimens, CLINICAL MICROBIOLOGY REVIEWS, Apr. 1990, p. 120-131; Collecting, preserving and shipping specimens for the diagnosis of avian influenza A(H5N1) virus infection, Guide for field operations, October 2006.

[00186] In some embodiments of any of the aspects, prior to amplification, target nucleic acids are isolated or purified from the sample. Nucleic acids can be isolated from a particular biological sample using any of a number of procedures, which are known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. For example, freeze-thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)). Non-limiting examples of methods for isolating/purifying nucleic acids from test samples include: (1) organic extraction, such as phenol-Guanidine Isothiocyanate (GITC)- based solutions (e.g., TRIZOL and TRI reagent); (2) silica-membrane based spin column technology (e.g., RNeasy and its variants); (3) paramagnetic particle technology (e.g., DYNABEADS mRNA DIRECT MICRO); (4) density gradient centrifugation using cesium chloride or cesium trifluoroacetate; (5) lithium chloride and urea isolation; (6) oligo(dt)- cellulose column chromatography; (7) non-column poly (A)+ purification/isolation; (8) organic extraction; (9) CHELEX 100 extraction; and (10) solid phase extraction.

[00187] In some embodiments of any of the aspects, the test sample can be an untreated test sample. As used herein, the phrase “untreated test sample” refers to a test sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments of any of the aspects, the test sample can be a frozen test sample. The frozen sample can be thawed before employing methods, assays and systems described herein. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein. In some embodiments of any of the aspects, the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments of any of the aspects, a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, homogenization, sonication, filtration, thawing, purification, and any combinations thereof. In some embodiments of any of the aspects, the test sample can be treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be employed, for example, to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. The skilled artisan is well aware of methods and processes appropriate for pre-processing of biological samples required for detection of a nucleic acid as described herein.

Reverse Transcription [00188] In some embodiments of any of the aspects, at least one of the target nucleic acid is an RNA and the method further comprises a step of reverse transcribing the RNA to a complementary DNA (cDNA) prior to or concurrently with the amplification step. The term “reverse transcriptase” (RT) refers to an RNA-dependent DNA polymerase used to generate complementary DNA (cDNA) from an RNA template. The cDNA can be single-stranded or double-stranded It is noted that the primer for reverse transcribing the RNA can be one of the primers used in the amplification step. For example, the primer for the reverse transcription can be same as the reverse primer in the amplification step.

Compositions

[00189] In another aspect, provided herein are compositions useful in detecting a plurality of target nucleic acids. The composition may comprise any of the components, reagents, assays, and systems discussed herein.

[00190] Generally, the composition comprises one or more of the following: (i) an exonuclease; (ii) a polymerase; (iii) a recombinase; (iv) single-stranded binding protein; (v) a first primer and optionally a second primer for amplification; (vi) one or more reagents for nucleic acid amplification; (vii) an amplified nucleic acid; (viii) one or more nucleic acid probes; (ix) one of more nucleic acid capture probes;(x) a ligand binding molecule capable of binding a capture ligand; (xi) a ligand binding molecule capable of binding a detection ligand; and/or (xii) a lateral flow strip. It is noted that a composition can comprise any one, two, three, four, five, six, seven, eight, nine, ten, eleven or all twelve of the components listed above. [00191] In some embodiments, the composition further comprises at least one of the following: detection reagents, a reverse transcriptase, reaction buffer, diluent, water, magnesium salt (such as magnesium acetate or magnesium chloride) dNTPs, reducing agent (such as DTT), a surfactant (such as SDS), and/or an RNase inhibitor.

Kits

[00192] Another aspect of the technology described herein relates to kits for detecting a plurality of target nucleic acid. The kit can comprise one or more of the following: (i) an exonuclease; (ii) a polymerase; (iii) a recombinase; (iv) single-stranded binding protein; (v) a first primer and optionally a second primer for amplification; (vi) one or more reagents for nucleic acid amplification; (vii) an amplified nucleic acid; (viii) one or more nucleic acid probes; (ix) one of more nucleic acid capture probes;(x) a ligand binding molecule capable of binding a capture ligand; (xi) a ligand binding molecule capable of binding a detection ligand; and/or (xii) a lateral flow strip. It is noted that a composition can comprise any one, two, three, four, five, six, seven, eight, nine, ten, eleven or all twelve of the components listed above. [00193] In some embodiments, the kit further comprises at least one of the following: detection reagents, a reverse transcriptase, reaction buffer, diluent, water, magnesium salt (such as magnesium acetate or magnesium chloride) dNTPs, reducing agent (such as DTT), a surfactant (such as SDS), and/or an RNase inhibitor.

[00194] In some embodiments of any of the aspects, the kit further comprises reagents for isolating nucleic acid from the sample. In some embodiments of any of the aspects, the kit further comprises reagents for isolating DNA from the sample. In some embodiments of any of the aspects, the kit further comprises reagents for isolating RNA from the sample. In some embodiments of any of the aspects, the kit further comprises detergent, e.g., for lysing the sample. In some embodiments of any of the aspects, the kit further comprises a sample collection device, such a swab. In some embodiments of any of the aspects, the kit further comprises a sample collection container, optionally containing transport media.

[00195] The components described herein can be provided singularly or in any combination as a kit. Such a kit includes the components described herein and packaging materials thereof. In addition, a kit optionally comprises informational material.

[00196] The kits can include, but are not limited to, any of the preprocessing reagents as described herein.

[00197] In some embodiments, the informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the aggregates for the methods described herein. For example, the informational material can describe methods for using the kits provided herein to perform an assay for detection of a target entity, e.g., a small molecule. The kit can also include an empty container and/or a delivery device, e.g., which can be used to deliver or prepare a test sample to a test container.

[00198] The informational material of the kits is not limited in its form. In many cases, the informational material, e.g, instructions, is provided in printed matter, e.g, a printed text, drawing, and/or photograph, e.g, a label or printed sheet. However, the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is a link or contact information, e.g, a physical address, email address, hyperlink, website, or telephone number, where a user of the kit can obtain substantive information about the formulation and/or its use in the methods described herein. Of course, the informational material can also be provided in any combination of formats. [00199] In some embodiments, the kit can contain separate containers, dividers or compartments for each component and informational material. For example, each different component can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, a collection of the magnetic particles is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label.

[00200] In some embodiments the kit includes a carrier for organizing and protecting the components in the kit during transport or storage. The carrier can be in any form including a bag, a box or a case, including handles, straps and wheels for convenient movement or storage.

Exemplary embodiments

[00201] Aspects of the disclosure can be described with the following Embodiments A-AF: [00202] Embodiment A: A method for multiplex detection of a plurality of target nucleic acids, the method comprising: (a) amplifying a plurality of target nucleic acids to produce a plurality of double-stranded amplicons, wherein a first strand of the double-stranded amplicons comprises at its 5’-terminal a nucleic acid modification capable of inhibiting 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease, wherein at least two members of the plurality are different from each other, and wherein said amplifying comprises recombinase polymerase amplification (RPA); (b) contacting the plurality of double-stranded amplicons from step (a) with a 5’ to 3’ exonuclease; to produce a plurality of single-stranded amplicons; (c) hybridizing the single-stranded amplicons with a first nucleic acid probe and a second nucleic acid probe to form a plurality of complexes, wherein the first nucleic acid probe comprises a nucleotide sequence substantially complementary to a first regions of a member of the plurality of single- stranded amplicons and the second nucleic acid probe comprises a nucleotide sequence substantially complementary to a second region of said member of the plurality, wherein each first nucleic acid probe independently comprises a detection ligand, and wherein each second nucleic acid probe independently comprises a capture ligand and wherein the capture ligand in a first complex of the plurality of complexes is distinguishable from the capture ligand in a second complex of the plurality of the complexes; and (d) detecting presence of the complexes in a lateral flow device, wherein the lateral flow device comprises: a first test region comprising a first ligand binding molecule capable of binding with the capture ligand in the first complex, and a second test region comprising a second ligand binding molecule capable of binding with the capture ligand in the second complex. [00203] Embodiment B: The method of Embodiment 1, wherein each capture ligand is independently selected from the group consisting of organic and inorganic molecules, peptides, polypeptides, proteins, peptidomimetics, glycoproteins, lectins, nucleosides, nucleotides, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, lipopolysaccharides, vitamins, steroids, hormones, cofactors, receptors, receptor ligands, and analogs and derivatives thereof.

[00204] Embodiment C: The method of Embodiment 1 or 2, wherein each capture ligand is an independently selected antigen.

[00205] Embodiment D: The method of any one of Embodiments A-C, wherein the ligand binding molecules capable of binding with the capture ligands independently are an antibody. [00206] Embodiment E: A method for multiplex detection of a plurality of target nucleic acids, the method comprising: (a) amplifying a plurality of target nucleic acids to produce a plurality of double-stranded amplicons, wherein one strand of the double-stranded amplicons comprises at its 5’-terminal a nucleic acid modification capable of inhibiting 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease, wherein at least two members of the plurality are different from each other, and wherein said amplifying comprises RPA; (b) contacting the plurality of double-stranded amplicons from step (a) with a 5’ to 3’ exonuclease; to produce a plurality of single-stranded amplicons; (c) hybridizing the single-stranded amplicons with a nucleic acid probe to form a plurality of complexes, wherein each nucleic acid probe independently comprises a detection ligand, and wherein a first nucleic acid probe comprises a nucleotide sequence substantially complementary to a first regions of a first member of the plurality of single-stranded amplicons and a second nucleic acid probe comprises a nucleotide sequence substantially complementary to a first region of a second member of the plurality of single- stranded amplicons; and (d) detecting presence of the complexes in a lateral flow device, wherein the lateral flow device comprises: a first test region comprising a first nucleic acid capture probe comprising a nucleotide sequence substantially complementary to a second region of the first member of the plurality of complexes; and a second test region comprising a second nucleic acid capture probe comprising a nucleotide sequence substantially complementary to a second region of the second member of the plurality of complexes. [00207] Embodiment F: The method of any one of Embodiments A-E, wherein the nucleic acid modification capable of inhibiting 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease is selected from the group consisting of modified internucleotide linkages, modified nucleobase, modified sugar, and any combinations thereof. [00208] Embodiment G: The method of any one of Embodiments A-F, wherein the first strand of the double-stranded amplicons independently comprises: (i) 1, 2, 3, 4, 5, 6 or more modified internucleotide linkages; (ii) an inverted nucleoside or 5’ to 5’ internucleotide linkage; (iii) a 2’-OH or a 2’-modified nucleoside; (iv) a 5’-modified nucleotide; (v) a 2' to 5’ linkage; (vi) an abasic nucleoside; (vii) an acyclic nucleoside; and (viii) any combinations of (i)-(vii).

[00209] Embodiment H: The method of any one of Embodiments A-G, wherein the first strand of the double-stranded amplicons independently comprises at least six phosphorothioate linkages in the 5’ -terminal.

[00210] Embodiment T The method of any one of Embodiments A-H, wherein the first strand of the double-stranded amplicons independently comprises at its 5’ -end a poly-dT nucleotide sequence comprising at least six nucleotides.

[00211] Embodiment J : The method of any one of Embodiments A-I, wherein the first strand of the double-stranded amplicons independently comprises at its 5’ -end a poly-dT nucleotide sequence comprising at least six nucleotides and at least six phosphorothioates.

[00212] Embodiment K: The method of any one of Embodiments A-J, wherein said detecting presence of the complexes comprises binding ligand binding molecule with the detection ligand and wherein the ligand binding molecule comprises a detectable label.

[00213] Embodiment L: The method of Embodiment K, wherein the detectable label is independently selected from the group consisting of a light-absorbing dye, a fluorescent dye, a luminescent or bioluminescent molecule, a quantum dot, a radiolabel, an enzyme, a calorimetric label.

[00214] Embodiment M: The method of Embodiment K or L, wherein the detectable label is selected from the group consisting of a light-absorbing dye, a fluorescent dye, a luminescent or bioluminescent molecule, a quantum dot, a radiolabel, an enzyme, a calorimetric label. [00215] Embodiment N: The method of any one of Embodiments K-M, wherein the detectable label is calorimetric label selected from the group consisting of colloidal gold, colored glass or plastic beads, and any combinations thereof.

[00216] Embodiment O: The method of Embodiment N, wherein the detectable label is a gold nanoparticle or a latex bead.

[00217] Embodiment P: The method of any one of Embodiments A-O, wherein each tag molecule independently is a detectable label.

[00218] Embodiment Q: The method of any one of Embodiments A-P, wherein each detection ligand is independently selected from the group consisting of a light-absorbing dye, a fluorescent dye, a luminescent or bioluminescent molecule, a quantum dot, a radiolabel, an enzyme, a calorimetric label.

[00219] Embodiment R: The method of any one of Embodiments A-Q, wherein each detection ligand is independently selected from the group consisting of a light-absorbing dye, a fluorescent dye, a luminescent or bioluminescent molecule, a quantum dot, a radiolabel, an enzyme, a calorimetric label.

[00220] Embodiment S: The method of any one of Embodiments A-R, wherein each detection ligand independently is a fluorescent dye.

[00221] Embodiment T: The method of any one of Embodiments A-S, wherein each detection ligand independently is fluorescein.

[00222] Embodiment U: The method of any one of Embodiments A-T, wherein the exonuclease is T7 exonuclease, lambda exonuclease, Exonuclease VIII, T5 exonuclease, or RecJf.

[00223] Embodiment V: The method of any one of Embodiments A-U, wherein at least one member of the plurality of target nucleic acids is single-stranded.

[00224] Embodiment W : The method of any one of Embodiments A-V, wherein at least one member of the plurality of target nucleic acids is double-stranded.

[00225] Embodiment X: The method of any one of Embodiments A-W, wherein at least one member of the plurality of target nucleic acids is RNA.

[00226] Embodiment Y : The method of any one of Embodiments A-X, wherein at least one member of the plurality of target nucleic acids is DNA.

[00227] Embodiment Z: The method of any one of Embodiments A-Y, wherein at least one member of the plurality of target nucleic acids is a viral nucleic acid.

[00228] Embodiment AA: The method of any one of Embodiments A-Z, wherein at least one member of the plurality of target nucleic acids is a first viral nucleic acid and at least one member of the plurality of target nucleic acids is a second viral nucleic acid.

[00229] Embodiment AB: The method of Embodiment AA, wherein the first and second viral nucleic acids are from different viruses.

[00230] Embodiment AC: The method of any one of Embodiments A-AB, wherein at least one member of the plurality of target nucleic acids is a viral RNA.

[00231] Embodiment AD: The method of any one of Embodiments A-AC, wherein at least one member of the plurality of target nucleic acids is a viral DNA. [00232] Embodiment AE: The method of any one of Embodiments A-AD, further comprising a step of heating the double-stranded amplicons prior to contacting with the exonuclease.

[00233] Embodiment AF: The method of any one of Embodiments A-AE, wherein the exonuclease is T7 exonuclease, lambda exonuclease, Exonuclease VIII, T5 exonuclease, or RecJf.

[00234] Aspects of the disclosure also can be described with the following Embodiments AG- AS:

[00235] Embodiment AG: A method for multiplex detection of target nucleic acids in a plurality of samples, the method comprising: (a) amplifying a target nucleic acid in each member of the plurality of samples to produce a double-stranded amplicon, wherein a first strand of the double-stranded amplicons comprises at its 5’ -terminal a detectable label and further comprises at its 3’-end a sample index domain, e.g., a domain/sequence for identifying the sample from which the amplified product came; (b) contacting the double-stranded amplicon from each member of the plurality of samples with a 5’ to 3’ exonuclease to produce a single-stranded amplicon for each member of the plurality of samples, wherein the single- stranded amplicon comprises at its 5’ -end the detectable label and further comprise at its 3’- end the sample index domain, e.g., a domain/sequence for identifying the sample from which the single-stranded or partially single-stranded amplicon came; (c) pooling the single-stranded or partially single-stranded amplicons from at least two members of the plurality of samples; and (d) detecting the pooled single-stranded amplicons in a lateral flow device, wherein the lateral flow device comprises: a plurality of capture/test regions, each capture/test region comprising a nucleic acid probe immobilized thereon, wherein the nucleic acid probe comprises a nucleotide sequence substantially complementary to at least a part of the index domain of a member of the pooled single-stranded amplicons, and wherein the immobilized nucleic acid probe of at least two capture/test regions are different.

[00236] Embodiment AH: The method of claim BB, wherein the exonuclease is T7 exonuclease, lambda exonuclease, Exonuclease VIII, T5 exonuclease, or RecJf.

[00237] The method of claim AG or AH, further comprising heating the amplified product prior to contacting with the exonuclease.

[00238] Embodiment AI: A method for multiplex detection of target nucleic acids in a plurality of samples, the method comprising: (a) amplifying a target nucleic acid in each member of the plurality of samples to produce a double-stranded amplicon, wherein a first strand of the double-stranded amplicon comprises at its 5’-terminal a detectable label and further comprises at its 3’-end a sample index domain, e.g., a domain/sequence for identifying the sample from which the amplified product came) and wherein a second strand of the double- stranded amplicon comprises at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) uridine nucleotide in a region complementary to the sample index domain; (b) contacting the amplified product from each member of the plurality of samples with a with Uracil-DNA glycosylase (UDG) to produce an amplicon having a single-stranded region (e.g., a partially single- stranded amplicon) for each member of the plurality of samples; (c) pooling the partially single-stranded amplicons from at least two members of the plurality of samples; and (d) detecting the pooled partially single-stranded amplicons in a lateral flow device, wherein the lateral flow device comprises: a plurality of capture/test regions, each capture/test region comprising a nucleic acid probe immobilized thereon, wherein the nucleic acid probe comprises a nucleotide sequence substantially complementary to at least a part of the single-stranded region of a member of the pooled partially single-stranded amplicons, and wherein the immobilized nucleic acid probe of at least two capture/test regions are different.

[00239] Embodiment AJ: The method of any one of Embodiments AG-AI, wherein the detectable label is independently selected from the group consisting of a light-absorbing dye, a fluorescent dye, a luminescent or bioluminescent molecule, a quantum dot, a radiolabel, an enzyme, a calorimetric label.

[00240] Embodiment AK: The method of any one of Embodiments AG-AJ, wherein the detectable label is selected from the group consisting of a light-absorbing dye, a fluorescent dye, a luminescent or bioluminescent molecule, a quantum dot, a radiolabel, an enzyme, a calorimetric label.

[00241] Embodiment AL: The method of any one of Embodiments AG-AK, wherein the detectable label is calorimetric label selected from the group consisting of colloidal gold, colored glass or plastic beads, and any combinations thereof.

[00242] Embodiment AM: The method of any one of Embodiments AG-AL, wherein the detectable label is a gold nanoparticle or a latex bead.

[00243] Embodiment AN: The method of any one of Embodiments AG- AM, wherein the method further comprises a step of reverse transcribing the target nucleic acid in at least one member of the plurality of samples.

[00244] Embodiment AO: The method of any one of Embodiments AG- AN, wherein said amplifying comprises isothermal amplification.

[00245] Embodiment AP: The method of any one of Embodiments AG-AO, wherein said amplifying comprises isothermal amplification selected from the group consisting of Recombinase Polymerase Amplification (RPA), Loop Mediated Isothermal Amplification (LAMP), Helicase-dependent isothermal DNA amplification (HDA), Rolling Circle Amplification (RCA), Nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR), polymerase Spiral Reaction (PSR), Hybridization Chain Reaction (HCR), Primer Exchange Reaction (PER), Signal Amplification by Exchange Reaction (SABER), transcription-based amplification system (TAS), Self-sustained sequence replication reaction (3 SR), Single primer isothermal amplification (SPIA), and cross-priming amplification (CPA).

[00246] Embodiment AQ: The method of any one of Embodiments AG-AP, wherein said amplifying comprises RPA.

[00247] Embodiment AR: A lateral flow device comprising: a lateral flow matrix, wherein the lateral flow matrix comprises: a plurality of capture zones, wherein each capture zone independently comprises a nucleic acid capture probe immobilized on the lateral flow matrix, wherein the capture probe is capable of hybridizing with a single-stranded or partially-single- stranded nucleic acid (e.g., a single-stranded or partially-single-stranded), and wherein the capture probe in at least one capture zone is different from the capture probe in at least one other capture zone.

[00248] Embodiment AS: The lateral flow device of Embodiment AR, wherein at least two of the capture zones are arranged in a predetermined pattern.

[00249] Aspects of the disclosure also can be described with the following numbered Embodiments AT-CV:

[00250] Embodiment AT : A method for multiplex detection of a plurality of target nucleic acids, the method comprising: (a) preparing single-stranded amplicons from a plurality of target nucleic acids, wherein at least two members of the plurality of target nucleic acids are different from each other; (b) detecting the single-stranded amplicons in a lateral flow device in the presence of a plurality of nucleic acid probes, wherein a first nucleic acid probe comprises a nucleotide sequence substantially complementary to a first region of a single-stranded amplicon and a second nucleic acid probe comprises a nucleotide sequence substantially complementary to a second region of said single-stranded amplicon, wherein each first nucleic acid probe independently comprises a detection ligand, and wherein each second nucleic acid probe independently comprises a capture ligand and wherein the capture ligand of a second nucleic acid probe complementary to a first single-stranded amplicon is different from the capture ligand of a second nucleic acid probe complementary to a second single-stranded amplicon, and wherein the lateral flow device comprises: a first test region comprising a first ligand binding molecule capable of binding with a first capture ligand, and a second test region comprising a second ligand binding molecule capable of binding with a second capture ligand, wherein the first and second capture ligands are different.

[00251] Embodiment AU: The method of Embodiment AU, wherein each capture ligand is independently selected from the group consisting of organic and inorganic molecules, peptides, polypeptides, proteins, peptidomimetics, glycoproteins, lectins, nucleosides, nucleotides, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, lipopolysaccharides, vitamins, steroids, hormones, cofactors, receptors, receptor ligands, and analogs and derivatives thereof.

[00252] Embodiment AV: The method of Embodiment AU, wherein each capture ligand is an independently selected antigen.

[00253] Embodiment AW: The method of any one of Embodiments AT-AV, wherein the ligand binding molecules capable of binding with the capture ligands independently are an antibody.

[00254] Embodiment AX: The method of any one of Embodiments AT -AW, wherein the single-stranded amplicons each comprise an index domain.

[00255] Embodiment AY: The method of any one of Embodiments AT-AX, wherein the index domain comprises a nucleotide sequence substantially complementary to the nucleic acid probe comprising the capture ligand.

[00256] Embodiment AZ: A method for multiplex detection of target nucleic acids in a plurality of samples, the method comprising: (a) preparing single-stranded amplicons from a plurality of target nucleic acids, wherein at least two members of the plurality of target nucleic acids are different from each other; (b) detecting the single-stranded amplicons in a lateral flow device in the presence of nucleic acid probes, wherein each nucleic acid probe independently comprises a detection ligand, and wherein a first nucleic acid probe comprises a nucleotide sequence substantially complementary to a first region of a first single-stranded amplicon and a second nucleic acid probe comprises a nucleotide sequence substantially complementary to a first region of a second single-stranded amplicon, and wherein the lateral flow device comprises: a plurality of capture/test regions, each capture/test region comprising a nucleic acid probe immobilized thereon, wherein the nucleic acid probe comprises a nucleotide sequence substantially complementary to at least a part of a single-stranded amplicon, and wherein the immobilized nucleic acid probe of at least two capture/test regions are different. [00257] Embodiment BA: A method for multiplex detection of target nucleic acids in a plurality of samples, the method comprising: (a) preparing single-stranded amplicons from a plurality of target nucleic acids, wherein at least two members of the plurality of target nucleic acids are different from each other, wherein each single-stranded amplicon comprises a detection ligand; (b) detecting the single-stranded amplicons in a lateral flow device, wherein the lateral flow device comprises: a plurality of capture/test regions, each capture/test region comprising a nucleic acid probe immobilized thereon, wherein the nucleic acid probe comprises a nucleotide sequence substantially complementary to at least a part of a single- stranded amplicon, and wherein the immobilized nucleic acid probes of at least two capture/test regions are different.

[00258] Embodiment BB: The method of Embodiment A Z or BA, wherein each single- stranded amplicon comprises an index domain.

[00259] Embodiment BC: The method of Embodiment BB, wherein at least a part of the index domain comprises a nucleotide sequence substantially complementary to a nucleic acid probe immobilized in a capture/test region of the lateral flow device.

[00260] Embodiment BD: The method of any one of Embodiments AT-BC, wherein the single-strand amplicons comprise at their 5’-terminus a nucleic acid modification capable of inhibiting 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease.

[00261] Embodiment BE: The method of Embodiment BD, wherein the nucleic acid modification capable of inhibiting 5’ to 3’ cleaving activity of a 5’ to 3’ exonuclease is selected from the group consisting of modified intemucleotide linkages, modified nucleobase, modified sugar, and any combinations thereof.

[00262] Embodiment BF: The method of any one of Embodiments AT -BE, wherein the single-stranded amplicons independently comprise: (i) 1, 2, 3, 4, 5, 6 or more modified intemucleotide linkages; (ii) an inverted nucleoside or 5’ to 5’ intemucleotide linkage; (iii) a 2' -OH or a 2' -modified nucleoside; (iv) a 5’ -modified nucleotide; (v) a 2' to 5’ linkage; (vi) an abasic nucleoside; (vii) an acyclic nucleoside; (viii) a spacer; (ix) left-handed DNA; (x) non- canonical nucleobases nucleotide; and (xi) any combinations of (i)-(x).

[00263] Embodiment BG: The method of any one of Embodiments AT-BF, wherein the single-stranded amplicon comprises at least six phosphorothioate linkages at the 5’ -terminus. [00264] Embodiment BH: The method of any one of Embodiment AT-BG, wherein at least one of the single-stranded amplicons comprises at its 5’ -end a poly-dT nucleotide sequence comprising at least six nucleotides.

[00265] Embodiment BE The method of any one of Embodiments AT-BH, wherein at least one of the single-stranded amplicons comprises at its 5’ -end a poly-dT nucleotide sequence comprising at least six nucleotides and at least six phosphorothioates. [00266] Embodiment BJ: The method of any one of Embodiments AT-BI, wherein said detecting comprises binding a ligand binding molecule with the detection ligand and wherein the ligand binding molecule comprises a detectable label.

[00267] Embodiment BK: The method of any one of Embodiments AT-BJ, wherein each detection ligand independently is a detectable label.

[00268] Embodiment BL: The method of Embodiment BJ or BK, wherein the detectable label is independently selected from the group consisting of a light-absorbing dye, a fluorescent dye, a luminescent or bioluminescent molecule, a quantum dot, a radiolabel, an enzyme, a calorimetric label.

[00269] Embodiment BM: The method of any one of Embodiments BJ-BL, wherein the detectable label is calorimetric label selected from the group consisting of colloidal gold, colored glass or plastic beads, and any combinations thereof.

[00270] Embodiment BN: The method of any one of Embodiments BJ-BM, wherein the detectable label is a gold nanoparticle or a latex bead.

[00271] Embodiment BO: The method of any one of Embodiments AT -BN, wherein each detection ligand independently is a fluorescent dye.

[00272] Embodiment BP: The method of any one of Embodiments AT -BO, wherein each detection ligand independently is fluorescein.

[00273] Embodiment BQ: The method of any one of Embodiments AT -BP, wherein the step of preparing the single-stranded amplicons comprises cleaving one strand of double-stranded amplicons prepared from the plurality of target nucleic acids.

[00274] Embodiment BR: The method of Embodiment BQ, wherein a first strand of the double-stranded amplicons comprises at its 5’ -terminus a nucleic acid modification capable of inhibiting 5’ to 3 cleaving activity of a 5’ to 3 exonuclease.

[00275] Embodiment BS: The method of Embodiment BR, wherein said cleaving one strand of the double-stranded amplicons comprises contacting the double-stranded amplicons with a 5’ to 3’ exonuclease.

[00276] Embodiment BT: The method of any one of Embodiments BQ-BS, further comprising a step of heating the double-stranded amplicons prior to contacting with the exonuclease.

[00277] Embodiment BU: The method of any one of Embodiments BQ-BT, wherein a first strand of the double-stranded amplicons comprises at least one uridine nucleotide.

[00278] Embodiment BV: The method of Embodiment BE!, wherein said uridine is in a region complementary to the index domain of the single-stranded amplicon. [00279] Embodiment BW: The method of any one of Embodiments BR-BV, wherein said cleaving one strand of the double-stranded amplicons comprises contacting the double- stranded amplicons with a Uracil-DNA glycosylase (UDG).

[00280] Embodiment BX: The method of any one of Embodiments AT-BW, wherein the method further comprises a step of amplifying the plurality of target nucleic acids.

[00281] Embodiment BY: The method of Embodiment BX, wherein said amplifying the plurality of target nucleic acids comprises isothermal amplification.

[00282] Embodiment BZ: The method of Embodiment BY, wherein said isothermal amplification is selected from the group consisting of: Recombinase Polymerase Amplification (RPA), Loop Mediated Isothermal Amplification (LAMP), Helicase-dependent isothermal DNA amplification (HD A), Rolling Circle Amplification (RCA), Nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR), polymerase Spiral Reaction (PSR), Hybridization Chain Reaction (HCR), Primer Exchange Reaction (PER), Signal Amplification by Exchange Reaction (SABER), transcription-based amplification system (TAS), Self-sustained sequence replication reaction (3 SR), Single primer isothermal amplification (SPIA), and cross-priming amplification (CPA).

[00283] Embodiment CA: The method of Embodiment BY or BZ, wherein said isothermal amplification is RPA.

[00284] Embodiment CB: The method of any one of Embodiments BX-CA, wherein a primer used in the amplification of a target nucleic acid comprises a nucleic acid modification capable of inhibiting 5 to 3 cleaving activity of a 5 to 3 exonuclease.

[00285] Embodiment CC: The method of any one of Embodiments BX-CB, wherein a primer used in the amplification of a target nucleic acid comprises a detection ligand.

[00286] Embodiment CD: The method of any one of Embodiment BX-CC, wherein a primer used in the amplification of the target nucleic acid comprises at least one uridine.

[00287] Embodiment CE: The method of any one of Embodiments AT-CD, wherein at least one member of the plurality of target nucleic acids is single-stranded.

[00288] Embodiment CF : The method of any one of Embodiments AT-CE, wherein at least one member of the plurality of target nucleic acids is double-stranded.

[00289] Embodiment CG: The method of any one of Embodiments AT-CF, wherein at least one member of the plurality of target nucleic acids is RNA.

[00290] Embodiment CH: The method of Embodiment CG, wherein the method further comprises reverse transcribing the RNA target nucleic acid. [00291] Embodiment Cl: The method of any one of Embodiments AT-CH, wherein at least one member of the plurality of target nucleic acids is DNA.

[00292] Embodiment CJ: The method of any one of Embodiments AT-CI, wherein at least one member of the plurality of target nucleic acids is a viral nucleic acid.

[00293] Embodiment CK: The method of any one of Embodiments AT-CJ, wherein at least one member of the plurality of target nucleic acids is a first viral nucleic acid and at least one member of the plurality of target nucleic acids is a second viral nucleic acid.

[00294] Embodiment CL: The method of Embodiment CK, wherein the first and second viral nucleic acids are from different viruses.

[00295] Embodiment CM: The method of any one of Embodiments AT-CL, wherein at least two capture zones of the lateral flow device are capable of detecting the same single-stranded amplicon.

[00296] Embodiment CN: The method of any one of Embodiments AT-CM, wherein at least two of the capture zones of the lateral flow device are arranged in a predetermined pattern. [00297] Embodiment CO: The method of any one of Embodiments AT-CN, further comprising a step of hybridizing the nucleic acid probes to the single-stranded amplicons prior to detection.

[00298] Embodiment CP: The method of any one of Embodiments AT-CO, further comprising a step of pooling the single-stranded amplicons from different target nucleic acids prior to detection.

[00299] Embodiment CQ: The method of any one of Embodiments AT-CP, wherein the single-stranded amplicons from different target nucleic acids are prepared simultaneously in one reaction vessel.

[00300] Embodiment CR: A lateral flow device comprising a lateral flow matrix, wherein the lateral flow matrix comprises a plurality of capture zones, wherein each capture zone independently comprises a capture moiety immobilized on the lateral flow matrix, and wherein: (i) at least two of the capture zones are configured to capture/detect the same target molecule; or (ii) at least one of the capture zone is configured to capture/detect a first target molecule and at least another one of the capture zone is configured to capture/detect a second target molecule, wherein the first and second target molecules are different.

[00301] Embodiment CS: The lateral flow device of Embodiment CR, wherein at least one of the capture zones is configured to capture/detect at least two different target molecules. [00302] Embodiment CT: The lateral flow device of Embodiment CR or CS, wherein at least two of the capture zones are arranged in a predetermined pattern. [00303] Embodiment CU: The lateral flow device of any one of Embodiments CR-CT, wherein the capture moiety is a nucleic acid.

[00304] Embodiment CV: The lateral flow device of any one of Embodiments CR-CU, wherein the capture moiety is a ligand binding molecule.

Some selected definitions

[00305] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[00306] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[00307] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder. [00308] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10- fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

[00309] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein. [00310] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of viral infection. A subject can be male or female.

[00311] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. a viral infection) or one or more complications related to such a condition, and optionally, have already undergone treatment for a viral infection or the one or more complications related to a viral infection. Alternatively, a subject can also be one who has not been previously diagnosed as having a viral infection or one or more complications related to a viral infection. For example, a subject can be one who exhibits one or more risk factors for a viral infection or one or more complications related to a viral infection or a subject who does not exhibit risk factors. A “subject in need” of testing for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[00312] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one component as described herein (e.g., sample, a target nucleic acid, target RNA, cDNA, amplification product, etc.). In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine. [00313] As used herein, the term “hybridizing”, “hybridize”, “hybridization”, “annealing”, or “anneal” are used interchangeably in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. In other words, the term “hybridization” refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide. The term “hybridization” may also refer to triple- stranded hybridization. The resulting (usually) double-stranded polynucleotide is a “hybrid” or “duplex.” In some embodiments of the various aspects described herein, the step of hybridizing comprises heating and/or cooling.

[00314] It is noted that the hybridization step can be carried out in the same reaction vessel used for preparing the amplified product. Alternatively, the amplified product can be isolated or purified from the amplification reaction prior to the hybridization step. In other words, the amplification step and the hybridization steps are in different reaction vessels.

[00315] “Hybridization conditions” will typically include salt concentrations of less than about 1 M, more usually less than about 500 mM and even more usually less than about 200 mM. Hybridization temperatures can be as low as 5° C., but are typically greater than 22° C., more typically greater than about 30° C., and often in excess of about 37° C. Hybridizations are usually performed under stringent conditions, i.e., conditions under which a probe will hybridize to its target subsequence. Stringent conditions are sequence-dependent and are different in different circumstances. Longer fragments may require higher hybridization temperatures for specific hybridization. As other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone. Generally, stringent conditions are selected to be about 5° C lower than the Tm for the specific sequence at a defined ionic strength and pH. Exemplary stringent conditions include salt concentration of at least 0.01 M to no more than 1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3 and a temperature of at least 25° C. For example, conditions of 5><SSPE (750 mM NaCl, 50 mM Na phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C are suitable for allele-specific probe hybridizations. For stringent conditions, see for example, Sambrook, Fritsche and Maniatis, Molecular Cloning A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press (1989) and Anderson Nucleic Acid Hybridization, 1st Ed., BIOS Scientific Publishers Limited (1999). “Hybridizing specifically to” or “specifically hybridizing to” or like expressions refer to the binding, duplexing, or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

[00316] The term “substantially identical” means two or more nucleotide sequences have at least 65%, 70%, 80%, 85%, 90%, 95%, or 97% identical nucleotides. In some embodiments, “substantially identical” means two or more nucleotide sequences have the same identical nucleotides.

[00317] The term “substantial complementary” or “substantially complementary” as used herein refers both to complete complementarity of binding nucleic acids, in some cases referred to as an identical sequence, as well as complementarity sufficient to achieve the desired binding of nucleic acids. Correspondingly, the term “complementary hybrids” encompasses substantially complementary hybrids.

[00318] As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50oC or 70oC for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

[00319] As used herein, the term “complementary,” in the context of an oligonucleotide (i.e., a sequence of nucleotides such as an oligonucleotide primers or a target nucleic acid) refers to standard Watson/Crick base pairing rules. For example, the sequence “5'-A-G-T-C- 3'” is complementary to the sequence “3 -T-C-A-G-5' ” Certain nucleotides not commonly found in natural nucleic acids or chemically synthesized may be included in the nucleic acids described herein; these include but not limited to base and sugar modified nucleosides, nucleotides, and nucleic acids, such as inosine, isocytosine and isoguanine. “Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson- Crick base pairs includes, but not limited to, G:U Wobble or Hoogsteen base pairing. In other words, complementarity need not be perfect; stable duplexes may contain mismatched base pairs, degenerative, or unmatched nucleotides. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, incidence of mismatched base pairs, ionic strength, other hybridization buffer components and conditions.

[00320] Complementarity may be partial in which only some of the nucleotide bases of two nucleic acid strands are matched according to the base pairing rules. Complementarity may be complete or total where all of the nucleotide bases of two nucleic acid strands are matched according to the base pairing rules. Complementarity may be absent where none of the nucleotide bases of two nucleic acid strands are matched according to the base pairing rules. In some embodiments of any of the aspects, two nucleic acid strands are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in detection methods that depend upon binding between nucleic acids. [00321] As used herein, the term “specific binding” refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a nontarget. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third non-target entity. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.

[00322] As used herein, the term “oligonucleotide” is intended to include, but is not limited to, a single-stranded DNA or RNA molecule, typically prepared by synthetic means. Nucleotides of the present invention will typically be the naturally-occurring nucleotides such as nucleotides derived from adenosine, guanosine, uridine, cytidine and thymidine. When oligonucleotides are referred to as “double-stranded,” it is understood by those of skill in the art that a pair of oligonucleotides exists in a hydrogen-bonded, helical array typically associated with, for example, DNA. In addition to the 100% complementary form of double-stranded oligonucleotides, the term “double-stranded” as used herein is also meant to include those form which include such structural features as bulges and loops (see Stryer, Biochemistry, Third Ed. (1988), incorporated herein by reference in its entirety for all purposes). [00323] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviations (2SD) or greater difference.

[00324] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%. In some embodiments of any of the aspects, the term “about” when used in connection with percentages can mean ±5%.

[00325] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[00326] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[00327] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[00328] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[00329] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[00330] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[00331] Other terms are defined herein within the description of the various aspects of the invention.

[00332] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[00333] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[00334] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[00335] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.